Universal donor stem cells and related methods

Abstract

To provide universal donor stem cells and related methods.SOLUTION: Disclosed herein are universal donor stem cells and related methods of their use and production. The universal donor stem cells disclosed herein are useful for overcoming the immune rejection in cell-based transplantation therapies. In certain embodiments, the universal donor stem cells disclosed herein do not express one or more MHC-I and MHC-II human leukocyte antigens. Similarly, in certain embodiments, the universal donor stem cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B and / or HLA-C) corresponding to MHC-I and MHC-II human leukocyte antigens, thereby rendering such cells hypoimmunogenic.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 158,999, filed May 8, 2015, the contents of which are incorporated by reference herein in their entirety. [Background technology]

[0002] government support This invention was made with Government support under DK097768 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 2. Background of the Invention Degenerative diseases pose a disproportionate threat to human health. Often with age, these diseases result in progressive deterioration of affected tissues and organs, ultimately resulting in disability and death of the affected subject. The promise of regenerative medicine is to replace diseased or missing cells with new healthy cells. In the past five years, a new paradigm in regenerative medicine has emerged - the use of human pluripotent stem cells (hPSCs) to generate any adult cell type for transplantation into patients. In principle, hPSC-based cell therapy has the potential to treat most, if not all, degenerative diseases; however, the success of such therapy may be limited by the subject's immune response.

[0004] Strategies being explored to overcome immune rejection include HLA matching (e.g., identical twin or umbilical cord banking), administering immunosuppressants to the subject, blocking antibodies, myelosuppression / mixed chimerism, HLA-matched stem cell repositories, and autologous stem cell therapy. There is a need for novel approaches, compositions and methods for overcoming immune rejection associated with cell replacement therapy. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein is an efficient strategy to overcome immune rejection in cell-based transplantation therapy by creating universal donor stem cell lines that form the basis of regenerative cell therapy as cell-based quality-controlled products.

[0006] The present inventors have succeeded in reducing or knocking out the expression of highly diverse classical MHC-I genes (HLA-A, HLA-B and HLA-C) and / or MHC-II genes in human pluripotent stem cells using genome editing tools such as TALEN and / or CRISPR systems.In certain embodiments, such reduction or knockout of the expression of MHC-I genes and / or MHC-II genes is achieved by directly and / or indirectly targeting the NLRC5 gene, the B2M gene and the CIITA gene as well as other components of the MHC enhanceosome (e.g., transcriptional regulators of MHC-I or MHC-II).

[0007] Disclosed herein is a method for preparing hypoimmunogenic stem cells, comprising modulating the expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens by stem cells, thereby preparing hypoimmunogenic stem cells.Also disclosed is a method for modulating the expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens by stem cells, comprising deleting one or more genes encoding one or more transcription regulators of MHC-I or MHC-II from at least one allele of the cell, thereby modulating the expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens.

[0008] In certain aspects, modulating the expression of one or more MHC-I and MHC-II human leukocyte antigens comprises reducing, inhibiting and / or disrupting the expression of one or more MHC-I and MHC-II human leukocyte antigens. In certain embodiments, modulating the expression of one or more MHC-I and MHC-II human leukocyte antigens comprises deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell. For example, in certain embodiments, such methods comprise deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II selected from the group consisting of NLRC5, CIITA, B2M and combinations thereof.

[0009] In certain aspects, the methods disclosed herein further comprise modulating the expression of one or more tolerogenic factors by the stem cells. Modulating the expression of a tolerogenic factor may comprise increasing the expression of the tolerogenic factor. Such a tolerogenic factor may be inserted into a safe harbor locus (e.g., the AAVS1 locus) of at least one allele of the cell. In certain embodiments, such a tolerogenic factor inhibits immune rejection. In certain embodiments, such a tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitor and IL-35.

[0010] Also disclosed herein is a method for preparing hypoimmunogenic stem cells, comprising modulating the expression of one or more tolerogenic factors by the stem cells, thereby preparing the hypoimmunogenic stem cells. In certain embodiments, modulating the expression of a tolerogenic factor comprises increasing the expression of the tolerogenic factor. Such a tolerogenic factor may be inserted into a safe harbor locus (e.g., the AAVS1 locus) of at least one allele of the cell. In certain embodiments, such a tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitor and IL-35. In certain embodiments, such a tolerogenic factor inhibits immune rejection of stem cells or differentiated stem cells prepared therefrom.

[0011] In certain aspects, the disclosed methods further comprise modulating expression of one or more MHC-I and MHC-II human leukocyte antigens by the cell, e.g., by deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell. In certain embodiments, the transcriptional regulators of MHC-I or MHC-II are selected from the group consisting of NLRC5, CIITA, B2M, and combinations thereof.

[0012] Human stem cells that do not express NLRC5 (e.g., NLRC5 - / - Also disclosed herein are human stem cells that do not express CIITA (e.g., knockout mutant stem cells). - / - Also disclosed are human stem cells that do not express B2M (e.g., knockout mutant stem cells). - / - Also disclosed are human stem cells that do not express one or more of NLRC5, CIITA and B2M. In certain embodiments, human stem cells that do not express one or more of HLA-A, HLA-B and HLA-C are also disclosed.

[0013] In certain embodiments, the stem cells disclosed herein express one or more tolerogenic factors (e.g., one or more tolerogenic factors that inhibit immune rejection). In certain aspects, such tolerogenic factors are inserted into a safe harbor locus (e.g., the AAVS1 locus) of at least one allele of the cell. In certain embodiments, the tolerogenic factors are selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitor and IL-35.

[0014] In some embodiments, the cells are embryonic stem cells. In certain embodiments, the cells are pluripotent stem cells. In certain embodiments, the stem cells are hypoimmunogenic.

[0015] In some embodiments, the cells have reduced expression of MHC-I compared to the original genotype or compared to wild-type human stem cells, and in certain embodiments, the cells have reduced expression of MHC-II compared to the original genotype or compared to wild-type human stem cells. For example, such cells have reduced expression of one or more of HLA-A, HLA-B and HLA-C compared to the original genotype or compared to wild-type human stem cells.

[0016] Also disclosed herein are human stem cells that have been modified to include one or more tolerogenic factors inserted into a safe harbor locus (e.g., the AAVS1 locus) of at least one allele of the cell. In certain embodiments, such tolerogenic factors inhibit immune rejection. In certain embodiments, the tolerogenic factors are selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitor, and IL-35. In certain aspects, such cells do not express one or more of NLRC5, CIITA, and B2M. In certain aspects, such cells do not express one or more of HLA-A, HLA-B, and HLA-C. In certain embodiments, such cells have reduced expression of one or more of HLA-A, HLA-B, and HLA-C compared to wild-type human stem cells.

[0017] Also disclosed herein is a method of using the hypoimmunogenic cells disclosed herein for cell replacement therapy.For example, the pluripotent stem cells disclosed herein can be incubated under appropriate conditions and differentiate into hypoimmunogenic cardiomyocytes, endothelial cells, hepatocytes or pancreatic β cells.

[0018] Also disclosed herein is a method for producing hypoimmunogenic stem cells, the method comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in the cell.

[0019] Also disclosed herein is a method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell.

[0020] Also disclosed herein is a method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in the cell.

[0021] Also disclosed herein is a method for producing hypoimmunogenic stem cells, the method comprising: (a) contacting a stem cell with a first pair of a Cas protein or a nucleic acid sequence encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in the cell; (b) contacting a stem cell with a second pair of a Cas protein or a nucleic acid sequence encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell; and / or (c) contacting a stem cell with a third pair of a Cas protein or a nucleic acid sequence encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in the cell.

[0022] Also disclosed is a method for producing hypoimmunogenic stem cells, the method comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in the cell.

[0023] Also disclosed is a method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell.

[0024] Also disclosed is a method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in the cell.

[0025] Also disclosed herein is a method for producing hypoimmunogenic stem cells, the method comprising: (a) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein, and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in the cell; (b) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein, and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell; and / or (c) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding a Cas protein, and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in the cell.

[0026] Also disclosed herein is a hypoimmunogenic stem cell comprising a modified genome, wherein the NLRC5 gene has been edited to reduce or eliminate NLRC5 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239.

[0027] Also disclosed herein is a hypoimmunogenic stem cell comprising a modified genome, wherein the CIITA gene has been edited to reduce or eliminate CIITA surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352.

[0028] Also disclosed herein are hypoimmunogenic stem cells comprising a modified genome, the hypoimmunogenic stem cells comprising a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cells by contacting the cells with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644.

[0029] A hypoimmunogenic stem cell comprising a modified genome, wherein (a) a first genome modification is edited in which the NLRC5 gene is edited to reduce or eliminate NLRC5 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353 to 81239; and (b) a first genome modification is edited in which the CIITA gene is edited to reduce or eliminate NLRC5 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352. Also disclosed herein are hypoimmunogenic stem cells comprising (a) a second genomic modification, wherein the B2M gene has been edited to reduce or eliminate CIITA surface expression and / or activity in the cell by contacting the cell with a ribonucleic acid encoding a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240 to 85644; and / or (b) a third genomic modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240 to 85644.

[0030] Also disclosed is a hypoimmunogenic stem cell comprising a modified genome, wherein the NLRC5 gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating NLRC5 surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239.

[0031] Also disclosed is a hypoimmunogenic stem cell comprising a modified genome, wherein the CIITA gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CIITA surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352.

[0032] Also disclosed are hypoimmunogenic stem cells comprising a modified genome, wherein the B2M gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating B2M surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644.

[0033] A hypoimmunogenic stem cell comprising a modified genome, comprising: (a) a first genomic modification in which the NLRC5 gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating NLRC5 surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239; (b) a first genomic modification in which the CIITA gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CIITA surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239; and / or (c) a B2M gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating B2M surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a first pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 5184-36352. Also disclosed herein are hypoimmunogenic stem cells comprising a third genomic modification, wherein the B2M gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating B2M surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a first pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 81240-85644.

[0034] The above-discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention. [Brief description of the drawings]

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0036] [Figure 1] Figure 1 shows the promise of regenerative medicine.

[0037] [Diagram 2] 2A-2B illustrate HLA barriers in transplantation. Figure 2A illustrates MHC-I and MHC-II surface molecules that define donor compatibility during organ transplantation. Figure 2B illustrates the HLA region 6p21.1-21.3.

[0038] [Diagram 3] Figures 3A-3B show targeting of transcriptional regulators of antigen presentation. Figure 3A shows a model of the MHC-II enhanceosome. Figure 3B shows a model of the NLRC5 enhanceosome in the MHC-I promoter.

[0039] [Figure 4] Figure 4 shows MHC-I expression in WT HuES9 and the indicated engineered mutant cell lines. Cells were stimulated with IFNγ for 48 hours and MHC-I expression was assessed by FACS.

[0040] [Diagram 5] 5A-5C show that CIITA-deficient stem cell-derived macrophages lack MHC-II expression. FIG. 5A depicts a timeline of macrophage differentiation. FIG. 5B depicts a bright field image of stem cell-derived macrophages in M-CSF medium on day 5. FIG. 5C depicts MHC-II expression in induced macrophages as assessed by qPCR 48 hours after stimulation with IFNγ, demonstrating reduced MHC-II expression compared to wild-type cells. As shown in FIG. 5C, MHC-II expression can be efficiently abrogated by targeting the first coding exon of the CIITA gene depicted in FIG. 10A-10B.

[0041] [Figure 6]FIG. 6 shows a timeline of the teratoma rejection study disclosed herein in which humanized NSG mouse-BLT mice are fed for three months to fully reconstitute the human immune system and then injected with the indicated genome-edited stem cell lines.

[0042] [Figure 7] Figure 7 demonstrates improved engraftment of genome-edited stem cells in humanized mice. Quantification of teratoma types observed following the teratoma rejection study depicted in Figure 6 is shown. Upon examination of the morphology of teratoma samples, distinct phenotypes were observed that correlated with the level of MHC-I expression.

[0043] [Figure 8] Figures 8A-8C demonstrate that proliferation of infiltrating CD8+ T cells indicates immune rejection. Figures 8A and 8B show immunostaining of teratoma-infiltrating CD3+ lymphocytes. Figure 8C shows co-staining for CD8 and Ki67, a proliferation marker, highlighting proliferating CD8+ T cells (white arrows). Nuclear DAPI staining is shown in blue.

[0044] [Figure 9] Figures 9A-9D demonstrate that deletion of NLRC5 using TALEN results in reduced MHC-I expression in induced pluripotent stem cell-derived hepatocyte-like cells (HLCs). Figure 9A shows the NLRC5 locus. Figure 9B shows targeting of the NLRC5 locus to suppress NLRC5 expression. Figures 9C-9D show reduced MHC-I expression in NLTC5- / - HLCs.

[0045] [Figure 10] Figures 10A-10B show targeting of the first coding exon of the CIITA gene. Figure 10A shows the CIITA locus. Figure 10B shows targeting of the CIITA locus to silence MHC-II expression.

[0046] [Figure 11]Figures 11A-11C demonstrate that tolerogenic factors such as PD-L1 and HLA-G can be expressed from safe harbor loci.

[0047] [Figure 12] 12 shows an exemplary amino acid sequence of a Cas protein. Yellow highlighting indicates the Ruv-C-like domain. Underlining indicates the HNH nuclease domain.

[0048] [Figure 13] FIG. 13 shows a comparison of MHC-II and MHC-I enhanceosomes.

[0049] [Figure 14A]Figures 14A-14J demonstrate the targeting of HLA-A, HLA-B and HLA-C. Figure 14A represents the HLA-B and HLA-C knockout strategy. Two short guide RNAs (sgRNAs) were designed upstream of the HLA-B locus and downstream of HLA-C (which allows for the excision of HLA-B and HLA-C genes). sgRNA#1 and sgRNA#2 target the HLA-B upstream region, while sgRNA#3 and sgRNA#4 target the HLA-C downstream region. Figure 14B shows that PCR screening confirmed clone 1D to be a homozygous knockout clone. Figure 14B includes a schematic showing the PCR validation strategy for successful deletion of HLA-B and HLA-C. Two pairs of wild-type (WT) primers flanking each cleavage site were designed, with predicted amplicon sizes of 545 bp and 472 bp. Clone 1D was identified as a homozygous knockout clone by the presence of a PCR band of approximately 680 bp generated with the KO primers and the absence of a band when two different sets of WT primers were used. Genomic DNA was isolated from the targeted HuES8 clones shown. Figure 14C provides a schematic showing sequence confirmation of HLA-B, HLA-C deletion in the 1D KO clone. Sequencing results of the 1D PCR products demonstrated successful deletion of HLA-B and HLA-C genes in HuES8. Figure 14D shows RT-PCR confirms loss of HLA-B and HLA-C mRNA expression in HuES8 cells. RT-PCR using HLA-B and HLA-C specific primers demonstrated the elimination of HLA-B and HLA-C mRNA expression in clone 1D. GAPDH, TOP1 and HPRT1 were used as internal controls. Figure 14E provides a schematic showing sequence confirmation of HLA-B RT-PCR bands obtained from WT and clone 1D. WT and 1D RT-PCR products amplified with HLA-B primers were sequenced and identified as HLA-B and HLA-A mRNA, respectively, using BLAST.These results demonstrated that in the absence of HLA-B gene, HLA-B specific primers amplify HLA-A mRNA in HuES8 clone 1D. Figure 14F shows karyotype analysis of HuES8 clone 1D by NanoString nCounter. HuES8 clone 1D shows normal karyotype as assessed by NanoString nCounter set. Figure 14G provides a schematic showing HLA-A knockout strategy using dual sgRNA approach. The schematic shows the location of two sgRNAs (sgRNA#5 and sgRNA#6) designed to bind upstream and downstream of HLA-A. Figure 14H shows on-target cleavage efficiency of HLA-A sgRNA using TIDE. Cleavage efficiency of sgRNA#5 and sgRNA#6 was determined in 293T cells using TIDE. Figure 14I demonstrates that PCR screening confirmed clone 4E as a heterozygous HLA-A knockout clone. A PCR screening strategy confirmed the deletion of HLA-A in HuES8. KO primers were designed with one primer annealing upstream and one primer annealing downstream of the cleavage site. With HLA-A deletion, the resulting amplicon is observed as 220 bp on a 2% agarose gel. Two pairs of WT primers were designed flanking each cleavage site with predicted amplicon sizes of 589 bp and 571 bp. Clone 4E was identified as a heterozygous clone by the presence of bands generated with KO and WT primers amplified from genomic DNA. Figure 14J includes a schematic showing that sequencing confirms successful deletion of HLA-A in HuES8 cells. Sequencing of PCR products amplified from genomic DNA of clone 4E using KO primers demonstrates successful deletion of HLA-A in HuES8. [Figure 14B] Same as above [Figure 14C] Same as above [Figure 14D] Same as above [Figure 14E] Same as above [Figure 14F] Same as above [Figure 14G] Same as above [Figure 14H] Same as above [Figure 14I] Same as above [Figure 14J] Same as above

[0050] [Figure 15A] Figure 15A-15D show TALEN-induced CIITA mutation and NLRC5 mutation in BJ-RIPSC cells and HuES9 cells. Figure 15A provides a schematic diagram of TALEN-induced CIITA mutation in BJ-RIPSC. Figure 15B provides a schematic diagram of TALEN-induced NLRC5 mutation in BJ-RIPSC. Figure 15C provides a schematic diagram of TALEN-induced NLRC5 mutation in HuES9. Figure 15D provides a schematic diagram of TALEN-induced CIITA mutation in HuES9. [Figure 15B] Same as above [Figure 15C] Same as above [Figure 15D] Same as above

[0051] [Figure 16A]Figures 16A-16G demonstrate targeting of NLRC5 and CIITA using the CRISPR system to achieve reduced MHC class I expression and complete loss of MHC class II expression. Figure 16A shows reduced MHC-I expression in NLRC5- / -Thp1 cells (including NLRC5 targeting strategy in Thp1 cells). + indicates 48 hours of IFNγ stimulation. Figure 16B provides a schematic of multiple targeting strategies. Figure 16B provides a schematic of targeting NLRC5 using CRISPR, a schematic of targeting B2M using CRISPR, a schematic of targeting CIITA using TALEN, and a schematic of targeting CIITA using CRISPR. Figure 16C shows reduced MHC class I expression in HuES9 cells after targeting with NLRC5 CRISPR or B2M CRISPR. The graph shows low basal MHC-I expression in stem cells. MHC-I expression can be increased by IFNγ stimulation. In IFNγ-treated NLRC5- / - cells, there is an approximately 50% reduction in MHC-I expression. In B2M- / - cells, a complete loss of MHC-I surface expression is shown. Figure 15D shows lentiviral transduction of Thp-1. A dual vector lentiviral GeCKO system is used. The two-component system includes Lenti-Cas9-Blasticidin (which acts to establish stable Thp-1 Cas9 cell lines) and Lenti-Guide-puro (which acts to transduce guides). Figure 16E shows various CRISPRs used for lentiviral transduction of Thp-1. CRISPRs targeting CIITA, NLRC5 and IRF1 are provided. Figure 16F shows that CIITA and NLRC5 act independently on MHC-II and MHC-I, respectively. Thp1 was transduced with lentiviruses encoding NLRC5 and CIITA. B2M CRISPR is used as a positive control. All cells were stimulated ON with 50U IFNγ to enhance HLA expression. HLA-A2 1:200; DR 1:100. Figure 16G shows that targeting IRF1 results in loss of MHC-II expression. Thp1 10 days after CRISPR transduction.All cells were stimulated ON with 50 U IFNγ. HLA-A2 1:200; DR 1:100. [Figure 16B] Same as above [Figure 16C] Same as above [Figure 16D] Same as above [Figure 16E] Same as above [Figure 16F] Same as above [Figure 16G] Same as above

[0052] [Figure 17A]Figures 17A-17K demonstrate targeting IRF1 utilizing the CRISPR system to achieve reduced MHC class I expression in human pluripotent stem cells (HuES9) and Thp-1 cells. Figure 17A shows a dual CRISPR strategy to target the IRF1 locus. Three different CRISPRs are shown. Figure 17B demonstrates testing of different IRF1 guide combinations. Screening results of different guide combinations are provided. Figure 17C shows a "dual guide strategy" for targeted deletion of IRF1. Screening results of the dual guide strategy are provided. Figure 17D provides a schematic showing sequence confirmation of IRF-1 CRISPR-induced deletion. Figure 17E provides screening results of IRF-1 targeting HuES9 cells. Screening is for the dual CRISPR strategy using CRISPR#2 and #3. The presence of PCR bands suggests successful targeting. Figure 17F demonstrates reconfirmation of IRF-1 clones. Screening results using primers KYM07 and TM377 are provided. Figure 17G demonstrates the genotype of IRF-1 clones. Screening results are provided. Figure 17H ​​provides a schematic showing sequence confirmation of IRF-1 CRISPR-induced deletion in clone 12. Figure 17I provides a schematic showing sequence confirmation of IRF-1 CRISPR-induced deletion in clone 17. Figure 17J provides a schematic showing sequence confirmation of IRF-1 CRISPR-induced deletion in clone 21. Figure 17K shows impaired MHC class I induction in IRF1- / - HuES9 clones after IFNγ treatment. P42 is WT; C7 is B2M KO; + indicates 48 hours of IFNγ treatment. [Figure 17B] Same as above [Figure 17C] Same as above [Figure 17D] Same as above [Figure 17E] Same as above [Figure 17F] Same as above [Figure 17G] Same as above [Figure 17H] Same as above [Figure 17I] Same as above [Figure 17J] Same as above [Figure 17K] Same as above

[0053] [Figure 18A] FIG. 18A-H demonstrate that targeting RFX5, RFX-ANK and RFX-AP in 293T cells using CRISPR results in reduced MHC class I expression. FIG. 18A demonstrates dual guide targeting of RFX5 in 293T cells. Four different CRISPR combinations were investigated. FIG. 18B shows that targeting RFX5 results in reduced MHC class I expression. FIG. 18C demonstrates dual guide targeting of RFX-ANK in 293T cells. Four different CRISPR combinations were investigated. FIG. 18D shows that targeting RFX-ANK results in reduced MHC class I expression. FIG. 18E shows that targeting RFX5 and RFX-ANK results in reduced MHC class I expression. FIG. 18F provides a bar graph showing that targeting RFX5 and RFX-ANK results in reduced MHC class I expression. Figure 18G demonstrates dual guide targeting of RFX-AP in 293T cells. Four different CRISPR combinations were investigated. Figure 18H shows that targeting of RFX-AP results in a reduction of MHC class I expression. [Figure 18B] Same as above [Figure 18C] Same as above [Figure 18D] Same as above [Figure 18E] Same as above [Figure 18F] Same as above [Figure 18G] Same as above [Figure 18H] Same as above

[0054] [Figure 19A]Figures 19A-19E demonstrate that targeting NFY-A, NFY-B and NFY-C using CRISPR results in reduced MHC class I expression. Figure 19A demonstrates dual guide targeting of NFY-A in 293T cells. Four different CRISPR combinations were investigated. Figure 19B demonstrates dual guide targeting of NFY-C in 293T cells. Four different CRISPR combinations were investigated. Figure 19C shows that targeting of NFY-C and NFY-A results in reduced MHC class I expression. Figure 19D demonstrates dual guide targeting of NFY-B in 293T cells. Four different CRISPR combinations were investigated. Figure 19E shows that targeting of NFY-B results in reduced MHC class I expression. [Figure 19B] Same as above [Figure 19C] Same as above [Figure 19D] Same as above [Figure 19E] Same as above

[0055] [Figure 20A] Figures 20A-20D show that surface transport of MHC class I molecules can be suppressed by disrupting the TAP1 gene (ER-resident peptide transporter). Figure 20A shows that TAP1 CRISPR expression reduces MHC-I surface expression in Jurkat T cells. Four different CRISPR combinations were investigated. Figure 20B demonstrates that TAP1 CRISPR expression reduces MHC-I surface expression in Jurkat T cells. Figure 20C demonstrates elimination of HLA surface expression in Jurkat (Cas9) T cells. The Jurkat cell line was established from the peripheral blood of a 14-year-old with acute T-cell leukemia by Schneider et al. Figure 20D shows elimination of HLA surface expression in Jurkat (Cas9) T cells. Cells were treated with IFNγ for 48 hours. [Figure 20B] Same as above [Figure 20C] Same as above [Figure 20D] Same as above

[0056] [Figure 21A] Figures 21A-21D demonstrate the use of a CRISPR gRNA identified as HLA Razor that allows for simultaneous deletion of all MHC class I alleles by targeting a conserved region in the HLA gene. Figure 21A provides a schematic showing targeting of a conserved sequence found in all HLAs by CRISPR or TALEN using HLA Razor. Two purple boxes indicate the binding sites of the panHLA TALEN pair. Blue arrows indicate the panHLA CRISPRs tested; the PAM is boxed in blue. Figure 21B shows expression of pan-HLA TALENs in 293T and HuES9 cells 72 hours after transfection. Figure 21C shows that HLA-Razor CRISPR blunts MHC class I expression. Cas9-GFP was co-transfected into 293T cells. Results are shown 72 hours after transfection. Figure 21D provides a schematic showing a comparison of the activity of two different HLA Razors. Two HLA Razor guides are shown. [Figure 21B] Same as above [Figure 21C] Same as above [Figure 21D] Same as above

[0057] [Figure 22A]Figure 22A-F show the results of the PD-L1 and HLA-G knock-in strategy. Figure 22A provides a schematic diagram showing the PD-L1 and HLA-G knock-in strategy. WT and knock-in (KI) primers for clone screening were designed. The amplicon with WT primers is predicted to be 488bp, and the amplicon with KI primers is predicted to be 403bp and 915bp. Figure 22B represents the design of the knock-in donor plasmid. The design of the knock-in donor plasmid shows that the reading frames of PD-L1 and HLA-G are linked by T2A and their expression is driven by the CAGGS promoter. Puromycin was used as a drug resistance marker after the SA-2A gene trap element. Figure 22C demonstrates ectopic PD-L1 and HLA-G expression in 293T cells. The expression of PD-L1 and HLA-G was investigated by FACS analysis in 293T cells transfected with the donor plasmid. APC-conjugated PD-L1 antibody and FITC-conjugated HLA-G antibody were used. Figure 22D shows ectopic HLA-G expression in JEG-3 cells. Donor plasmids were transfected into HLA-G- / -JEG-3 cell line, and ectopic HLA-G expression was investigated by FACS analysis 48 hours after transfection. PE-conjugated HLA-G antibody (MEM / G9) was used to detect surface HLA-G surface expression. Figure 22E provides PCR screening results confirming clone 1G as a heterozygous KI clone of PD-L1 / HLA-G. Clone 1G was identified as a heterozygous KI clone by the presence of bands amplified from genomic DNA of HuES8 cells targeted using both WT and KI-specific primers. Figure 22F shows stable PD-L1 and HLA-G expression from safe harbor locus in HuES8. PD-L1 expression was verified in HuES8 KI clone 1G by FACS analysis using APC-conjugated PD-L1 antibody. [Figure 22B] Same as above [Figure 22C] Same as above [Figure 22D] Same as above [Figure 22E] Same as above [Figure 22F] Same as above

[0058] [Figure 23] Figure 23 demonstrates the expression of CD47 in 293T cells and hPSCs using a knock-in strategy. Human CD47 was cloned into an expression plasmid driven by a CAG promoter, which further contains an IRES-GFP. 293T cells already express high levels of CD47 (human "don't eat me" signal), which prevents phagocytosis of cells by macrophages. In both 293T cells and human pluripotent stem cells (HuES9), expression can be increased by overexpression of CD47, as detected by FACS using a CD47-specific antibody. Overexpression of CD47 supports the engraftment of stem cell-derived transplants by protecting cells from macrophage phagocytosis.

[0059] [Figure 24] Figure 24 demonstrates that deletion of TRAC and TRBC in HuES9 disrupts TCR expression. Using a dual guide RNA approach, deletions were introduced into the TRAC and TRBC loci in HuES9 cells. The TCRA wt band is 249bp, and after deletion, it is 209bp. The TCRB wt band is 162bp, and after deletion, it is 140bp. The bands marked with * are TCRB KO in HuES9 cells; the bands marked with ** are TCRA KO in HuES9 cells; the bands marked with *** are TCRA KO in HuES9 B2M- / -CIITA- / - cells. The TCRA KO in HuES9 B2M- / -CIITA- / - cells is a triple knockout stem cell line for B2M- / -, CIITA- / - and TCR- / -. Upon differentiation into T cells, this triple knockout stem cell line lacks MHC-I, MHC-II and TCR surface expression.

[0060] [Figure 25A]Figures 25A-25D demonstrate targeting of CD274 / B7-H1 / PD-L1 in various cell lines. Figure 25A shows examples of CRISPR guide sequences that can be used when targeting CD274 / B7-H1 / PD-L1. CD274 / B7-H1 / PD-L1 knockout cell lines are shown. Figure 25B shows screening of targeted B7-H1 colonies in JEG-3 cells. Screening revealed a CRISPR cleavage efficiency of 22 / 265, or about 8.3%. Figure 25C demonstrates reconfirmation of the 501 melanoma knockout clone. Figure 25D demonstrates reconfirmation of the MalMe melanoma knockout clone. [Figure 25B] Same as above [Figure 25C] Same as above [Figure 25D] Same as above

[0061] [Figure 26A]Figures 26A-26P demonstrate the targeting of co-inhibitory / co-stimulatory receptors or their ligands. For each target, the four CRISPRs shown were cloned and tested for on-target activity in 293T cells. The expected size of the PCR bands when cleavage of both CRISPRs occurs is shown below the gel image. Figure 26A shows co-stimulatory / co-inhibitory molecules and their receptors on T cells. Figure 26B demonstrates dual guide targeting of TIGIT in 293T cells. All four CRISPRs were found to be functional. Figure 26C demonstrates dual guide targeting of TIM3 in 293T cells. All four CRISPRs were found to be functional. Figure 26D demonstrates dual guide targeting of HVEM in 293T cells. All four CRISPRs were found to be functional. Figure 26E demonstrates dual guide targeting of 2B4 / CD244 in 293T cells. All four CRISPRs were found to function. Figure 26F demonstrates the dual guide targeting of CD28 in 293T cells. CRISPR#1, #2 and #3 were found to function. Figure 26G demonstrates the dual guide targeting of OX40 in 293T cells. CRISPR#1, #2 and #4 were found to function. Figure 26H demonstrates the dual guide targeting of B71 in 293T cells. CRISPR#1, #3 and #4 were found to function. Figure 26I demonstrates the dual guide targeting of CD226 in 293T cells. CRISPR#1 and #2 were found to function. Figure 26J demonstrates the dual guide targeting of CD2 in 293T cells. CRISPR#1, #2 and #3 were found to function. Figure 26K demonstrates the dual guide targeting of LAG3 in 293T cells. CRISPR#2 and #3 were found to function. Figure 26L demonstrates the dual guide targeting of BTLA in 293T cells. All four CRISPRs were found to function. Figure 26M demonstrates the dual guide targeting of ICOS in 293T cells. CRISPR#2, #3 and #4 were found to function.Figure 26N demonstrates the dual guide targeting of CD27 in 293T cells. CRISPR#1 and #2 are found to function. Figure 26O demonstrates the dual guide targeting of ST2 in 293T cells. All four CRISPRs are found to function. Figure 26P demonstrates the dual guide targeting of GITR in 293T cells. CRISPR#1 and #3 are found to function. [Figure 26B] Same as above [Figure 26C] Same as above [Figure 26D] Same as above [Figure 26E] Same as above [Figure 26F] Same as above [Figure 26G] Same as above [Fig. 26H] Same as above [Figure 26I] Same as above [Figure 26J] Same as above [Figure 26K] Same as above [Figure 26L] Same as above [Figure 26M] Same as above [Figure 26N] Same as above [Figure 26O] Same as above [Figure 26P] Same as above

[0062] [Figure 27] Figure 27 shows the various cell lines tested and the targets on which the cell lines were tested. HuES8 and HuES9 are human ES cell lines. BJ-RiPSC is an iPSC line. All other enhanceosome components not shown in the table (e.g., RFX and NFY) were tested only in HEK293T cells.

[0063] [Figure 28A]Figures 28A-28J provide that modified ES cells can differentiate into a variety of different cell types with reduced or absent HLA expression. Figure 28A provides that modified ES cells can differentiate into mesenchymal progenitor cells (MPCs), endothelial cells (ECs), macrophages, hepatocytes, beta cells, and neural progenitor cells (NPCs). Figure 28B shows reduced MHC-I expression in NLRC5- / - human ES cells. In stem cells, basal MHC-I expression is low. Expression can be increased by IFNγ stimulation. In IFNγ-treated NLRC5- / - cells, approximately 50% reduction in MHC-I expression occurs. Figure 28C shows reduced MHC-I expression in NLRC5- / - human mesenchymal progenitor cells (MPCs). A comparison of HuES9 cells and MPCs is provided. Figure 28D shows reduced MHC-I expression in stem cell-derived NLRC5- / - endothelial cells (ECs). FIG. 28E demonstrates that ECs show similar differentiation efficiency. FIG. 28F shows loss of HLA expression in B2M- / -CIITA- / -ECs. N=3, 48 hours IFNγ treatment. FIG. 28G demonstrates reduced MHC class I expression in NLRC5- / - hepatocyte-like cells. Schematic demonstrating the targeting strategy and CRISPR design is included. At day 7 of terminal hepatocyte differentiation, HLCs are generated from BJ-RiPSCs. FIG. 28H demonstrates that mutation of CIITA abrogates MHC class II expression in hESC-derived macrophages. Schematic demonstrating the targeting strategy and CRISPR design is included. HuES9-derived iMФ, M-CSF at day 5. FIG. 28I shows neural progenitor cell (NPC) differentiation. B2M- / -CITIA- / -HuES9 form nestin+ neural rosettes (white arrows). Figure 28J demonstrates the adaptation of stem cells to spin culture for β-cell differentiation, which is the first step in the β-cell differentiation protocol. [Figure 28B] Same as above [Figure 28C] Same as above [Figure 28D] Same as above [Figure 28E] Same as above [Figure 28F] Same as above [Figure 28G] Same as above [Fig. 28H] Same as above [Figure 28I] Same as above [Figure 28J] Same as above

[0064] [Figure 29A] Figures 29A-29B provide in vivo data in a teratoma model. Figure 29A provides that "low immunogenic" DKO cell lines were generated. The generated cell lines include WT HuES9, NLRC5- / -CIITA- / -HuES9, and B2M- / -CIITA- / -HuES9. The WT cell line showed expression of MHC-I and MHC-II. The NLRC5- / -CIITA- / - cell line showed reduced MHC-I expression and no MHC-II expression. The B2M- / -CIITA- / - cell line showed no expression of MHC-I and MHC-II. Figure 29B shows improved engraftment of genome-edited stem cells in humanized mice. The three cell lines tested were WT HuES9, NLRC5- / -CIITA- / -HuES9, and B2M- / -CIITA- / -HuES9. [Figure 29B] Same as above

[0065] [Figure 30A] Figures 30A-30D demonstrate CRISPR targeting of B7-H3 in JEG-3 cells. Figure 30A provides a CRISPR guide sequence for targeting B7-H3. Figure 30B shows screening of targeted B7-H3 colonies in JEG-3 cells. CRISPR cutting efficiency is shown to be 15 / 80, or about 18.7%. Figure 30C shows confirmation of B7-H3 knockout by sequencing. Figure 30D demonstrates loss of B7-H3 surface expression in targeted JEG3 clones. [Figure 30B] Same as above [Figure 30C] Same as above [Figure 30D] Same as above

[0066] [Diagram 31]Figure 31 shows the loss of MHC class I surface expression in B2M- / - Jurkat Cas9 T cells compared to Jurkat Cas9 T cells that do not contain a knockout of B2M.

[0067] [Diagram 32] Figures 32A-32H demonstrate targeting of clinically relevant loci in human cells using CRISPR / Cas9. Figure 32A is a schematic of gRNAs targeting B2M. Figure 32B is a histogram of B2M surface expression in HEK293T cells. Figure 32C shows B2M deletion efficiency in HEK293T cells using various gRNAs; n=3 (mean ± SEM). Figure 32D is a schematic of gRNAs targeting CCR5. Orange and green arrows represent primer pairs used to amplify the analyzed region. Figure 32E shows the results of a Surveyor assay for each gRNA targeting CCR5 in K562 cells. % indels are shown below each guide. Figure 32F shows B2M deletion efficiency of selected gRNAs in primary CD4+ T cells compared to 293T cells; n=6 (mean ± SEM). Figure 32G shows the results of Surveyor assay of crCCR5_A and crCCR5_B targeting CCR5 in K562 cells and HSPCs. Figure 32H shows the clonal deletion efficiency of crCCR5_A and crCCR5_B targeting CCR5 in HSPCs (n=2) as determined by Sanger sequencing (Note: crB2M_14 is not represented in the schematic in panel A as it is located 20Kb downstream of the coding sequence).

[0068] [Diagram 33]Figures 33A-33E demonstrate the evaluation of on-target mutation efficiency of various gRNAs targeting B2M. Figure 33A shows the B2M deletion efficiency for all gRNAs targeting the B2M locus in HEK293T cells as measured by flow cytometry. Pooled data of three independent experiments are shown as mean ± SEM. Figure 33B shows the B2M deletion efficiency of selected guides in HEK293T cells as measured by CEL Surveyor assay as % indels. Figure 33C is a comparison of B2M surface expression in HEK293T cells and primary CD4+ T cells when transfected with Cas9 and guide crB2M_13. Figure 33D shows the B2M deletion efficiency of selected guides targeting the B2M locus in primary CD4+ T cells as measured by flow cytometry. FIG. 33E shows the B2M deletion efficiency of the selection guides in primary CD4+ T cells, measured as % indels by CEL Surveyor assay.

[0069] [Diagram 34]Figures 34A-34E depict a dual gRNA approach for CRISPR / Cas9 genome editing in primary human hematopoietic stem cells and effector cells. Figure 34A is a schematic of the dual gRNA approach for targeting the B2M locus. gRNA pairs are in red. Base pair offset between Cas9 sites for each gRNA combination (right panel). Figure 34B shows B2M deletion efficiency in CD4+ T cells for six dual gRNA combinations (n=3; mean ± SEM). Figure 34C is a FACS plot showing loss of B2M expression of either crB2M_13 or crB2M_8 alone or in combination in primary CD4+ T cells. Figure 34D is a schematic of the dual gRNA approach for targeting CCR5. gRNA pairs are in red. Orange and green arrowheads represent primer pairs used to amplify the region. Offset between Cas9 sites for each gRNA pair (right panel). Figure 34E is a gel electrophoresis image of CD34+ HSPC-derived clones targeted with crCCR5_D+Q analyzed by PCR. Note the deletion of the 205 bp region between the two gRNA cleavage sites (upper panel; WT: wild type; ΔCCR5: deletion; green* represents WT clones; orange* represents heterozygous clones; red* represents homozygous deletion clones). Clonal deletion efficiency for three dual gRNA combinations targeting CCR5 in CD34+ HSPCs (n=4; % mean ± SEM; lower panel).

[0070] [Diagram 35]Figures 35A-35G demonstrate the targeting efficiency of dual gRNA combinations. Figure 35A shows the B2M deletion efficiency for six dual gRNA combinations from three independent donors as measured by flow cytometry. Figure 35B is a FACS plot showing the loss of MHC class I surface expression (lower panel) after B2M deletion (upper panel). Figure 35C is a schematic of the single-cell nested PCR strategy for the B2M locus (left panel). Black and grey arrowheads: control primer pairs, orange and green arrowheads: primer pairs flanking the targeted region. %B2M null single cells are shown (right panel, n=301). Figure 35D is a Sanger sequencing chromatogram showing the predicted deletion of the targeted region at the B2M locus. Figure 35E shows the clonal CCR5 deletion efficiency for three dual gRNA combinations in CD34+HSPC-mPB obtained from multiple donors. DNA isolated from individual colonies was analyzed by PCR and gel electrophoresis. Figure 35F is a schematic of the single-cell nested PCR strategy to determine CCR5 deletion in primary CD4+ T cells (left panel). % CCR5 null single cells are shown (right panel, n=363). Figure 35G shows the Sanger sequencing chromatogram showing the predicted deletion in the targeted region.

[0071] [Diagram 36] Figure 36 demonstrates the B2M deletion efficiency of the selection guide in 293T cells. The arrows in the Surveyor assay indicate the nuclease cleavage junctions.

[0072] [Figure 37] FIG. 37 demonstrates a comparison of B2M surface expression in 293T cells when transfected with AsCpf1 and the guide crB2M.

[0073] [Figure 38] FIG. 38 demonstrates a comparison of B2M surface expression in 293T cells when transfected with LbCpf1 and guide crB2M.

[0074] [Figure 39] Figure 39 represents Cpf1 crRNA design and cloning information.

[0075] [Figure 40A] Figures 40A-40G demonstrate the generation and characterization of B2M KO JEG3 cells using TALENs. Figure 40A depicts the B2M TALEN design and induced mutations. Figure 40B depicts the analysis of B2M at the transcript and protein levels. Figure 40C demonstrates the analysis of B2M at the surface expression level. Figure 40D demonstrates that ΔB2M clones lack MHC-I surface expression. Figure 40E demonstrates that ΔB2M clones lack HLA-G surface expression. Figure 40F demonstrates that ΔB2M clones lack HLA-C surface expression. Figure 40G demonstrates that ΔB2M clones lack HLA-E surface expression. [Figure 40B] Same as above [Figure 40C] Same as above [Figure 40D] Same as above [Figure 40E] Same as above [Diagram 40F] Same as above [Figure 40G] Same as above

[0076] [Diagram 41] Figure 41 demonstrates an exemplary TCR targeting strategy of the present invention. Figure 41 is a schematic showing the location of CRISPR gRNAs targeting the first coding exons of the TCR alpha and beta chains, respectively.

[0077] [Diagram 42]Figures 42A-B demonstrate T cell receptor deletion in Jurkat T cells. Figure 42A shows the results of FACS analysis using anti-CD3 antibody revealing successful TCR deletion in Jurkat T cells stably expressing Cas9 nuclease. Figure 42B shows the results of a SURVEYOR™ assay confirming truncation at the TCRa and TCRb loci.

[0078] [Diagram 43] Figures 43A-B demonstrate TCR deletion in primary human CD3+ T cells. Figure 43A shows the results of a SURVEYOR™ assay demonstrating CRISPR cleavage at the TCRa and TCRb loci in CD3+ T cells from two independent donors. Figure 43B shows the loss of TCR surface expression demonstrated by FACS analysis.

[0079] [Diagram 44] Figures 44A-C demonstrate an exemplary PD-1 locus targeting strategy of the invention. Figure 44A is a schematic of the PD-1 targeting strategy. Figure 44B demonstrates that the dual CRISPR strategy results in truncation by both CRISPRs targeting the PD-1 locus in HEK293T cells. Figure 44C is a schematic of sequencing that confirmed the predicted deletion at the PD-1 locus following transfection of two CRISPRs targeting the PD-1 gene (PDCD1).

[0080] [Diagram 45] Figures 45-35B demonstrate loss of PD-1 expression in Jurkat T cells. Figure 45A shows the results of a FACS analysis demonstrating loss of PD-1 expression in activated Jurkat T cells. Figure 45B shows the results of a SURVEYOR™ assay confirming truncation at the PD-1 locus.

[0081] [Figure 46]Figure 46A-C demonstrate an exemplary CTLA4 locus targeting strategy of the present invention. Figure 46A is a schematic diagram of the CTLA4 targeting strategy. Figure 46B demonstrates that the dual CRISPR strategy results in a cut by both CRISPRs targeting the CTLA4 locus in HEK293T cells. Figure 46C is a schematic diagram of sequencing that confirmed the predicted deletion at the CTLA4 locus after transfection of two CRISPRs targeting the CTLA4 gene (CTLA4).

[0082] [Figure 47] Figure 47A and Figure 47B demonstrate cleavage at the CTLA-4 locus in Jurkat T cells. Figure 47A demonstrates that the dual CRISPR strategy results in cleavage by both CRISPRs targeting the CTLA4 locus in Jurkat T cells. Figure 47B shows the results of a SURVEYOR™ assay demonstrating successful cleavage by both CTLA4 CRISPRs in Jurkat T cells.

[0083] The following Tables 8-55 are submitted herewith as Exhibits 1-47, respectively: Table 8 - Exemplary gRNA sequences useful for targeting HLA-A; Table 9 - Exemplary gRNA sequences useful for targeting HLA-B; Table 10 - Exemplary gRNA sequences useful for targeting HLA-C; Table 11 - Exemplary gRNA sequences useful for targeting RFX-ANK; Table 12 - Exemplary gRNA sequences useful for targeting CIITA; Table 13 - Exemplary gRNA sequences useful for targeting NFY-A; Table 14 - Exemplary gRNA sequences useful for targeting NLRC5; Table 15 - Exemplary gRNA sequences useful for targeting B2M; Table 16 - Exemplary gRNA sequences useful for targeting RFX5; Table 17 - Exemplary gRNA sequences useful for targeting RFX-AP; Table 18 - Exemplary gRNA sequences useful for targeting HLA-G; Table 19 - Exemplary gRNA sequences useful for targeting HLA-E; Table 20 - Exemplary gRNA sequences useful for targeting NFY-B; Table 21 - Exemplary gRNA sequences useful for targeting PD-L1; Table 22 - Exemplary gRNA sequences useful for targeting NFY-C; Table 23 - Exemplary gRNA sequences useful for targeting IRF1; Table 24 - Exemplary gRNA sequences useful for targeting TAP1; Table 25 - Exemplary gRNA sequences useful for targeting GITR; Tables 26-4. Exemplary gRNA sequences useful for targeting 1BB; Table 27 - Exemplary gRNA sequences useful for targeting CD28; Table 28 - Exemplary gRNA sequences useful for targeting B7-1; Table 29 - Exemplary gRNA sequences useful for targeting CD47; Table 30 - Exemplary gRNA sequences useful for targeting B7-2; Table 31 - Exemplary gRNA sequences useful for targeting OX40; Table 32 - Exemplary gRNA sequences useful for targeting CD27; Table 33 - Exemplary gRNA sequences useful for targeting HVEM; Table 34 - Exemplary gRNA sequences useful for targeting SLAM; Table 35 - Exemplary gRNA sequences useful for targeting CD226; Table 36 - Exemplary gRNA sequences useful for targeting ICOS; Table 37 - Exemplary gRNA sequences useful for targeting LAG3; Table 38 - Exemplary gRNA sequences useful for targeting TIGIT; Table 39 - Exemplary gRNA sequences useful for targeting TIM3; Table 40 - Exemplary gRNA sequences useful for targeting CD160; Table 41 - Exemplary gRNA sequences useful for targeting BTLA; Table 42 - Exemplary gRNA sequences useful for targeting CD244; Table 43 - Exemplary gRNA sequences useful for targeting LFA-1; Table 44 - Exemplary gRNA sequences useful for targeting ST2; Table 45 - Exemplary gRNA sequences useful for targeting HLA-F; Table 46 - Exemplary gRNA sequences useful for targeting CD30; Table 47 - Exemplary gRNA sequences useful for targeting B7-H3; Table 48 - Exemplary gRNA sequences useful for targeting VISTA; Table 49 - Exemplary gRNA sequences useful for targeting TLT; Table 50 - Exemplary gRNA sequences useful for targeting PD-L2; Table 51 - Exemplary gRNA sequences useful for targeting CD58; Table 52 - Exemplary gRNA sequences useful for targeting CD58; Table 53 - Exemplary gRNA sequences useful for targeting CD2; and Table 54 - Exemplary gRNA sequences useful for targeting HELIOS.

[0084] Exhibits 1-47 (Tables 8-54, respectively) are specifically incorporated herein by reference: Attachment 1 (File name: Table8.txt; Creation date: May 9, 2016; File size: 223,026 bytes); Attachment 2 (File name: Table9.txt; Creation date: May 9, 2016; File size: 327,895 bytes); Attachment 3 (File name: Table10.txt; Creation date: May 9, 2016; File size: 280,849 bytes); Attachment 4 (File name: Table11.txt; Creation date: May 9, 2016; File size: 998,046 bytes); Attachment 5 (File name: Table12.txt; Creation date: May 9, 2016; File size: 4,717,823 bytes); Attachment 6 (File name: Table13.txt; Creation date: May 9, 2016; File size: 2,813,407 bytes); Attachment 7 (File name: Table14.txt; Creation date: May 9, 2016; File size: 6,568,742 bytes); Attachment 8 (File name: Table15.txt; Creation date: May 9, 2016; File size: 728,685 bytes); Attachment 9 (File name: Table16.txt; Creation date: May 9, 2016; File size: 766,106 bytes); Attachment 10 (File name: Table17.txt; Creation date: May 9, 2016; File size: 874,435 bytes); Attachment 11 (File name: Table18.txt; Creation date: May 9, 2016; File size: 232,536 bytes); Attachment 12 (File name: Table19.txt; Creation date: May 9, 2016; File size: 539,932 bytes); Attachment 13 (File name: Table20.txt; Creation date: May 9, 2016; File size: 2,256,084 bytes); Attachment 14 (File name: Table21.txt; Creation date: May 9, 2016; File size: 1,303,081 bytes); Attachment 15 (File name: Table22.txt; Creation date: May 9, 2016; File size: 6,821,299 bytes); Attachment 16 (File name: Table23.txt; Creation date: May 9, 2016; File size: 1,095,386 bytes); Attachment 17 (File name: Table24.txt; Creation date: May 9, 2016; File size: 941,281 bytes); Attachment 18 (File name: Table25.txt; Creation date: May 9, 2016; File size: 378,368 bytes); Attachment 19 (File name: Table26.txt; Creation date: May 9, 2016; File size: 2,706,509 bytes); Attachment 20 (File name: Table27.txt; Creation date: May 9, 2016; File size: 3,578,977 bytes); Attachment 21 (File name: Table28.txt; Creation date: May 9, 2016; File size: 3,638,039 bytes); Attachment 22 (File name: Table29.txt; Creation date: May 9, 2016; File size: 5,083,645 bytes); Attachment 23 (File name: Table30.txt; Creation date: May 9, 2016; File size: 4,481,092 bytes); Attachment 24 (File name: Table31.txt; Creation date: May 9, 2016; File size: 393,567 bytes); Attachment 25 (File name: Table32.txt; Creation date: May 9, 2016; File size: 675,503 bytes); Attachment 26 (File name: Table33.txt; Creation date: May 9, 2016; File size: 1,002,258 bytes); Attachment 27 (File name: Table34.txt; Creation date: May 9, 2016; File size: 454,540 bytes); Attachment 28 (File name: Table35.txt; Creation date: May 9, 2016; File size: 10,463,522 bytes); Attachment 29 (File name: Table36.txt; Creation date: May 9, 2016; File size: 2,755,910 bytes); Attachment 30 (File name: Table37.txt; Creation date: May 9, 2016; File size: 733,191 bytes); Attachment 31 (File name: Table38.txt; Creation date: May 9, 2016; File size: 1,674,331 bytes); Attachment 32 (File name: Table39.txt; Creation date: May 9, 2016; File size: 2,622,419 bytes); Attachment 33 (File name: Table40.txt; Creation date: May 9, 2016; File size: 2,049,613 bytes); Attachment 34 (File name: Table41.txt; Creation date: May 9, 2016; File size: 4,016,813 bytes); Attachment 35 (File name: Table42.txt; Creation date: May 9, 2016; File size: 4,177,884 bytes); Attachment 36 (File name: Table43.txt; Creation date: May 9, 2016; File size: 4,326,759 bytes); Attachment 37 (File name: Table44.txt; Creation date: May 9, 2016; File size: 1,007,674 bytes); Attachment 38 (File name: Table45.txt; Creation date: May 9, 2016; File size: 1,690,144 bytes); Attachment 39 (File name: Table46.txt; Creation date: May 9, 2016; File size: 5,427,706 bytes); Attachment 40 (File name: Table47.txt; Creation date: May 9, 2016; File size: 3,510,941 bytes); Attachment 41 (File name: Table48.txt; Creation date: May 9, 2016; File size: 3,043,135 bytes); Attachment 42 (File name: Table49.txt; Creation date: May 9, 2016; File size: 274,899,026 bytes); Attachment 43 (File name: Table50.txt; Creation date: May 9, 2016; File size: 7,273,026 bytes); Attachment 44 (File name: Table51.txt; Creation date: May 9, 2016; File size: 2,336,524 bytes); Attachment 45 (File name: Table52.txt; Creation date: May 9, 2016; File size: 274,899,026 bytes); Attachment 46 (File name: Table53.txt; Creation date: May 9, 2016; File size: 1,820,108 bytes); Attachment 47 (Filename: Table54.txt; Creation Date: May 9, 2016; File Size: 17,165,777 bytes). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Detailed Description of the Invention Recent advances in stem cell biology have made it possible to contemplate using a subject's own cells as an unlimited source for transplantation, as generally depicted in, for example, FIG. 1. Unfortunately, genome editing and generation of induced pluripotent stem cells (iPSCs) and subsequent differentiation of such iPSCs are still costly and time-consuming processes with high variability in terms of pluripotency, epigenetic state, differentiation capacity and genome stability. It has also been found that the changes that occur during genome editing and long-term culture trigger adaptive immune responses, resulting in immune rejection even of autologous stem cell-derived transplants. To overcome the problem of immune rejection of subjects by stem cell-derived transplants, the inventors have developed and herein disclosed universal donor stem cells that represent a viable source for any transplantable cell type. Advantageously, the universal stem cells disclosed herein are not rejected by the immune system of recipient subjects, regardless of the genetic makeup of the subject.

[0086] The invention disclosed herein uses genome editing technology (e.g., CRISPR / Cas system or TALEN system) to reduce or eliminate the expression of important immune genes in human ES cells and iPSCs (e.g., by deleting genomic DNA of important immune genes), or in certain cases inserting tolerance-inducing factors, to make them and differentiated cells prepared therefrom less immunogenic and less susceptible to immune rejection by the subject into which such cells are transplanted. When used herein to characterize cells, the term "low immunogenicity" generally means that such cells are less susceptible to immune rejection by the subject into which such cells are transplanted. For example, compared to unmodified wild-type cells, such low immunogenic cells can be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less susceptible to immune rejection by the subject into which such cells are transplanted. In some embodiments, genome editing techniques (e.g., CRISPR / Cas or TALEN systems) are used to modulate (e.g., reduce or eliminate) expression of MHC-I and MHC-II genes.

[0087] In certain embodiments, the invention disclosed herein relates to stem cells whose genomes have been modified to reduce or delete key components of HLA expression. Similarly, in certain embodiments, the invention disclosed herein relates to stem cells whose genomes have been modified to insert one or more tolerance-inducing factors. The present invention contemplates altering target polynucleotide sequences in any manner available to those skilled in the art, for example, by utilizing TALEN or CRISPR / Cas systems. Such CRISPR / Cas systems may use a variety of Cas proteins (Haft et al., PLoS Comput Biol. 2005;1(6)e60). In some embodiments, the CRISPR / Cas system is a type I CRISPR system. In some embodiments, the CRISPR / Cas system is a type II CRISPR system. In some embodiments, the CRISPR / Cas system is a type V CRISPR system. Although examples of methods utilizing CRISPR / Cas (e.g., Cas9 and Cpf1) and TALEN are described in detail herein, it should be understood that the present invention is not limited to the use of these methods / systems.Other methods known to those skilled in the art for targeting polynucleotide sequences to reduce or eliminate expression in target cells can be utilized herein.

[0088] The present invention contemplates changing, e.g., modifying or truncating, target polynucleotide sequences in cells for any purpose, but in particular, to reduce or eliminate the expression or activity of the encoded product.For example, the CRISPR / Cas system can be used to target transcriptional regulators of antigen presentation to produce hypoimmunogenic stem cells. In some embodiments, a target polynucleotide sequence in a cell (e.g., an ES cell or an iPSC) is altered to produce a mutant cell. As used herein, a "mutated cell" generally refers to a cell whose resulting genotype differs from its original genotype or wild-type cell. In some cases, for example, when a normally functioning stem gene is altered using a CRISPR / Cas system, a "mutated cell" exhibits a mutant phenotype. In some embodiments, a target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, a target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0089] In some embodiments, the change is an indel. As used herein, "indel" refers to a mutation resulting from insertion, deletion, or a combination thereof. As those skilled in the art will recognize, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the change is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one nucleotide. The CRISPR / Cas system can be used to induce any length of indel or point mutation in a target polynucleotide sequence.

[0090] In some embodiments, the change results in the knockout of the target polynucleotide sequence or a part thereof.For example, the knockout of the target polynucleotide sequence in cells can be carried out in vitro, in vivo or ex vivo for both therapeutic and research purposes.The knockout of the target polynucleotide sequence in cells can be useful for treating or preventing disorders associated with the expression of the target polynucleotide sequence (for example, by knocking out the mutant allele in cells ex vivo and introducing the cells containing the knocked out mutant allele into a subject).

[0091] As used herein, "knockout" includes the deletion of all or a portion of a target polynucleotide sequence in a manner that disrupts the function of the target polynucleotide sequence or its expression product.

[0092] In some embodiments, the change results in a reduction in the expression of the target polynucleotide sequence. The terms "reduce", "reduced", "reduction" and "reduction" are all used herein generally to refer to a statistically significant amount of reduction. However, to avoid any doubt, "reduced", "reduced", "reduction" and "reduction" include a reduction of at least 10% compared to the reference level, such as at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or up to 100% (inclusive) reduction (i.e., non-existent levels compared to the reference sample), or any reduction between 10 and 100% compared to the reference level.

[0093] The terms "increased," "increase" or "enhance" or "activate" are all used herein generally to mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased," "increase" or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to and including 100%, or any increase of 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase of 2-fold to 10-fold or more compared to a reference level.

[0094] The term "statistically significant" or "significantly" refers to statistical significance, and generally means a concentration of a marker that is two standard deviations (2SD) below (i.e., lower) than normal. The term refers to statistical evidence that there is a difference. It is defined as the probability of deciding to reject the null hypothesis when the null hypothesis is in fact correct. The decision is often made using a p-value.

[0095] In some embodiments, the alteration is a homozygous alteration. In some embodiments, the alteration is a heterozygous alteration.

[0096] In some embodiments, the change results in the correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. The CRISPR / Cas system can be used to correct any kind of mutation or error in the target polynucleotide sequence. For example, the CRISPR / Cas system can be used to insert a nucleotide sequence that is missing from the target polynucleotide sequence by deletion. The CRISPR / Cas system can also be used to delete or excise a nucleotide sequence from the target polynucleotide sequence by insertion mutation. In some cases, the CRISPR / Cas system can be used to replace an incorrect nucleotide sequence with a correct nucleotide sequence (e.g., to restore a function to the target polynucleotide sequence that is impaired by a loss-of-function mutation).

[0097] The CRISPR / Cas system can alter target polynucleotides with surprisingly high efficiency. In certain embodiments, the efficiency of alteration is at least about 5%. In certain embodiments, the efficiency of alteration is at least about 10%. In certain embodiments, the efficiency of alteration is about 10% to about 80%. In certain embodiments, the efficiency of alteration is about 30% to about 80%. In certain embodiments, the efficiency of alteration is about 50% to about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 85%. In some embodiments, the efficiency of alteration is greater than or equal to about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%. In some embodiments, the efficiency of alteration is equal to about 100%.

[0098] In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

[0099] In some embodiments, the CRISPR / Cas system of the present invention comprises a Cas protein or a nucleic acid sequence encoding the Cas protein, and at least one to two ribonucleic acids (e.g., gRNAs) capable of orienting and hybridizing the Cas protein to a target motif of a target polynucleotide sequence. In some embodiments, the CRISPR / Cas system comprises a Cas protein or a nucleic acid sequence encoding the Cas protein, and at least one pair of ribonucleic acids (e.g., gRNAs) capable of orienting and hybridizing a single ribonucleic acid or Cas protein to a target motif of a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues linked by peptide bonds (i.e., a polymer of amino acids), including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants and analogs of the above.

[0100] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitutions and / or modifications may prevent or reduce proteolysis and / or extend the half-life of the polypeptide in a cell. In some embodiments, the Cas protein may comprise peptide bond substitutions (e.g., ureas, thioureas, carbamates, sulfonylureas, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise alternative amino acids (e.g., D-amino acids, β-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications including moieties (e.g., PEGylation, glycosylation, lipidation, acetylation, terminal capping, etc.).

[0101] In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. In some embodiments, the Cas protein comprises an E. coli subtype Cas protein (also known as CASS2). Exemplary E. coli subtype Cas proteins include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Exemplary Ypest subtype Cas proteins include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises an Nmeni subtype Cas protein (also known as CASS4). Exemplary Nmeni subtype Cas proteins include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

[0102] In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Staphylococcus aureus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus thermophilic Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Neisseria meningitides Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Treponema denticola Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof from any bacterial species. The Cas9 protein is typically a member of a type II CRISPR system that includes a trans-coding small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc) and a Cas protein. The Cas9 protein (also known as CRISPR-associated endonuclease Cas9 / Csn1) is a polypeptide that includes 1368 amino acids. An exemplary amino acid sequence of the Cas9 protein (SEQ ID NO:1) is shown in Figure 12. Cas9 contains two endonuclease domains, including a RuvC-like domain (residues 7-22, 759-766, and 982-989) that cleaves target DNA non-complementary to the crRNA, and an HNH nuclease domain (residues 810-872) that cleaves target DNA complementary to the crRNA. In Figure 12, the RuvC-like domain is highlighted in yellow and the HNH nuclease domain is underlined.

[0103] In some embodiments, the Cas protein is a Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is a Cpf1 or a functional portion thereof from any bacterial species. In some aspects, the Cpf1 is a Francisella novicida U112 protein or a functional portion thereof. In some aspects, the Cpf1 is an Acidaminococcus sp. BV3L6 protein or a functional portion thereof. In some aspects, the Cpf1 is a Lachnospiraceae bacterium ND2006 protein or a functional portion thereof. The Cpf1 protein is a member of the V-type CRISPR system. The Cpf1 protein is a polypeptide comprising about 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Cpf1 uses a single ribonuclease domain to cleave target DNA in a staggered pattern. The staggered DNA double-strand break results in a 4 or 5 nt 5' overhang.

[0104] As used herein, a "functional portion" refers to a portion of a peptide that retains the ability to complex with at least one ribonucleic acid (e.g., a guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Cpf1 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a HNH nuclease domain. In some embodiments, the functional portion of the Cpf1 protein comprises a functional portion of a RuvC-like domain.

[0105] It should be appreciated that the present invention contemplates various methods of contacting a target polynucleotide sequence with a Cas protein (e.g., Cas9). In some embodiments, an exogenous Cas protein may be introduced into a cell in a polypeptide form. In certain embodiments, a Cas protein may be conjugated or fused to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates the uptake of a molecule into a cell. A cell-penetrating polypeptide may contain a detectable label.

[0106] In certain embodiments, the Cas protein may be conjugated or fused to a charged protein (e.g., having a positive, negative, or total neutral charge). Such linkage may be covalent. In some embodiments, the Cas protein may be fused to a super positively charged GFP to significantly increase the cell penetration ability of the Cas protein (Cronican et al., ACS Chem Biol. 2010;5(8):747-52). In certain embodiments, the Cas protein may be fused to a protein transduction domain (PTD) to facilitate cell entry. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositive GFP. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a cell penetrating peptide. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a PTD. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a tat domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to an oligoarginine domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a penetratin domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a superpositive GFP.

[0107] In some embodiments, the Cas protein may be introduced into a cell containing a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein (e.g., Cas9 or Cpf1). The process of introducing the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA as described herein. In some embodiments, the nucleic acid comprises an mRNA. In some embodiments, the nucleic acid comprises a modified mRNA as described herein (e.g., a synthetic modified mRNA).

[0108] In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one or two ribonucleic acids are introduced into the cell via viral transduction (e.g., lentiviral transduction).

[0109] In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA) described herein.

[0110] The methods of the invention contemplate the use of any ribonucleic acid that can orient and hybridize a Cas protein to a target motif of a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises a tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises a CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that orients and hybridizes a Cas protein to a target motif of a target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that orients and hybridizes a Cas protein to a target motif of a target polynucleotide sequence in a cell. In some embodiments, the one to two ribonucleic acids both comprise a guide RNA that orients and hybridizes a Cas protein to a target motif of a target polynucleotide sequence in a cell. As one of skill in the art will recognize, the ribonucleic acids of the invention can be selected to hybridize to a variety of different target motifs depending on the particular CRISPR / Cas system and sequence of the target polynucleotide used. The one to two ribonucleic acids may also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif that is immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein. In some embodiments, the one to two ribonucleic acids are each designed to hybridize to a target motif that is immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein (which is adjacent to a mutant allele located between the target motifs).

[0111] In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 2 to 817976. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 2 to 817976.

[0112] In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 2 to 817976. In some embodiments, at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 2 to 817976.

[0113] In some embodiments, the one to two ribonucleic acids each comprise a guide RNA that directs the Cas protein to a target motif of a targeted polynucleotide sequence in a cell and hybridizes thereto. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to a sequence on the same strand of the targeted polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to a sequence on the opposite strand of the targeted polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are not complementary to and / or hybridize to a sequence on the opposite strand of the targeted polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to overlapping target motifs of the targeted polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to an offset target motif of a targeted polynucleotide sequence.

[0114] In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides. In some embodiments, the target motif is at least 20 nucleotides in length. In some embodiments, the target motif is a DNA sequence of 20 nucleotides. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides and immediately precedes an NRG motif. In some aspects, the NRG motif is NGG or NAG. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NGG motif recognized by a Cas protein. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NAG motif recognized by a Cas protein. In some embodiments, the target motif is a DNA sequence of 20 nucleotides beginning with a G and immediately precedes an NGG motif recognized by a Cas protein. In some embodiments, the target motif is G(N)19NGG. In some embodiments, the target motif is (N)20NGG.

[0115] In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides and immediately precedes an NNGRRT motif. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NNGRRT motif. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides and immediately precedes an NNNRRT motif. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NNNRRT motif. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NNNRRT motif. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides and immediately precedes an NNAGAAW motif. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NNAGAAW motif. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides and immediately precedes an NNNNGATT motif. In some embodiments, the target motif is a DNA sequence of 20 nucleotides and immediately precedes an NNNNGATT motif. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides and immediately precedes an NAAAAC motif. In some embodiments, the target motif is a 20 nucleotide DNA sequence immediately preceding the NAAAAC motif. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence having a 5' T-rich region (e.g., a TTTN motif). In some embodiments, the target motif is a 20 nucleotide DNA sequence having a 5' T-rich region (e.g., a TTTN motif).

[0116] In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides immediately preceding the NRG (e.g., NGG or NAG) motif recognized by the S. pyogenes Cas protein. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides immediately preceding the NNGRRT motif recognized by the S. aureus Cas protein. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides immediately preceding the NNNRRT motif recognized by the S. aureus Cas protein. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides immediately preceding the NNAGAAW motif recognized by the S. thermophilus Cas protein. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides immediately preceding the NNNNGATT motif recognized by the N. meningitides Cas protein. In some embodiments, the target motif is a DNA sequence of 17-23 nucleotides immediately preceding the NAAAAC motif recognized by the T. denticola Cas protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence having a 5' T-rich region (e.g., a TTTN motif) recognized by the Cpf1 protein of Acidaminococcus or Lachnospiraceae.

[0117] In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in the cell. Those skilled in the art will recognize that a variety of techniques can be used to select an appropriate target motif to minimize off-target effects (e.g., bioinformatics analysis). In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif that is immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein. In some embodiments, each of the one to two ribonucleic acids is designed to hybridize to a target motif that is immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein (which is adjacent to a mutant allele located between the target motifs).

[0118] In some aspects, the targeted polynucleotide sequence in the cell is modified using a gene editing system (e.g., TALEN, CRISPR / Cas, etc.) to reduce or eliminate the expression and / or activity of one or more important immune genes in the cell. In some embodiments, the present disclosure provides that the targeted polynucleotide sequence in the cell is modified (e.g., using a CRISPR / Cas system) to delete a continuous stretch of genomic DNA from one or both alleles of the cell (e.g., to delete one or more important immune genes). In some embodiments, the targeted polynucleotide sequence in the cell is modified (e.g., using a CRISPR / Cas system) to insert a genetic mutation into one or both alleles of the cell. In yet other embodiments, the pluripotent stem cells disclosed herein may be subjected to complementary genome editing approaches (e.g., using CRISPR / Cas systems) whereby such stem cells are modified to delete both contiguous stretches of genomic DNA (e.g., important immune genes) from one or both alleles of the cells and to insert one or more tolerance-inducing factors, such as HLA-G or PD-L1, into one or both alleles of the cells to locally suppress the immune system and improve transplant survival.

[0119] To evaluate HLA expression and to evaluate the immunogenicity of pluripotent stem cell line, for example in preclinical humanized mouse model, the pluripotent stem cell disclosed herein can be differentiated into related cell types.For example, the pluripotent stem cell disclosed herein can be incubated under appropriate conditions and differentiated into mesenchymal progenitor cell (MPC), low immunogenic cardiomyocyte, endothelial cell (EC), macrophage, hepatocyte, β cell (e.g., pancreatic β cell) or neural progenitor cell (NPC).

[0120] The pluripotent stem cells and methods disclosed herein have a profound impact on regenerative medicine by paving the way for rigorously tested pluripotent donor stem cell lines that can grow and differentiate into a very large number of cells, are widely available to all medical institutions, and can be used on demand to treat patients suffering from degenerative diseases, thereby eliminating the need to use the patient's own cells as a source for autologous transplantation on a case-by-case basis.In addition, the resulting cell products are protected from immune attack, so they represent a new form of treatment for autoimmune diseases (e.g., MS (multiple sclerosis) and diabetes) where autologous cells are still susceptible to immune attack.However, immune-privileged pluripotent donor stem cell-derived cell products are protected from autoimmune rejection.

[0121] The pluripotent stem cells disclosed herein can be used, for example, to diagnose, monitor, treat and / or cure the presence or progression of a disease or condition in a subject. As used herein, "subject" means a human or an animal. In certain embodiments, the subject is a human. In certain embodiments, the subject is an adolescent. In certain embodiments, the subject is treated in vivo, in vitro and / or in utero. In certain aspects, the subject in need of treatment by the methods disclosed herein has a condition or is suspected or at high risk of developing such a condition.

[0122] As depicted in FIG. 2A, HLA represents an immunological barrier to successful transplantation of stem cells or differentiated stem cells in a subject. Novel compositions, cells and related methods useful for overcoming the HLA immunological barrier to transplantation are disclosed herein. As depicted in FIG. 2B, the major histocompatibility complex (MHC) is a genetic locus on human chromosome 6p21 that encodes a highly polymorphic gene family of surface molecules that define donor compatibility during organ transplantation. MHC class I (MHC-I) and MHC class II (MHC-II) play a key role in activating adaptive immune responses by presenting antigens to T lymphocytes. A comparison of the MHC-II enhanceosome and the MHC-I enhanceosome is provided in FIG. 13. Humans have three classical MHC-Ia molecules (HLA-A, HLA-B and HLA-C) that are essential for the detection and elimination of viruses, cancerous cells and transplanted cells. In addition, there are three non-classical MHC-Ib molecules (HLA-E, HLA-F and HLA-G) that have immune-regulatory functions. In certain situations, such as cell-based transplantation therapy, MHCs perform important cellular functions, but they can also contribute to immune rejection. Provided herein are novel cells, compositions and methods that are useful for addressing such HLA-based immune rejection of transplanted cells. knock out

[0123] In certain embodiments, the invention disclosed herein relates to genomic modification of one or more targeted polynucleotide sequences of stem cell genome that regulates expression of MHC-I and / or MHC-II. In some embodiments, gene editing systems are used to modify one or more targeted polynucleotide sequences. In some embodiments, CRISPR / Cas systems are used to delete one or more targeted polynucleotide sequences. MHC-I molecules are composed of MHC-encoded heavy chains and the invariant subunit β2-microglobulin (B2M). Antigen-derived peptides are presented to CD8 T cells bearing antigen-specific T cell receptors by the MHC-I-B2M complex on the cell surface. Peptides are mainly produced from the degradation of cytoplasmic proteins by specialized proteasomes or immunoproteasomes optimized to generate MHC class I peptides, which contain several IFN-γ-inducible subunits. Unlike MHC-II, which is found primarily in antigen-presenting cells, MHC-Ia is ubiquitously expressed in nearly all nucleated cells (Pamer et al., Annu Rev Immunol (1998) 16:323-358.) Both MHC-I and MHC-II genes are highly inducible by IFN-γ stimulation.

[0124] Efficient removal of the HLA barrier can be achieved by one or more of the following: (1) direct targeting of polymorphic HLA alleles (HLA-A, -B, -C) and MHC-II genes; (2) removal of B2M, which prevents surface trafficking of all MHC-I molecules; and / or (3) deletion of components of the MHC enhanceosome that are important for HLA expression, such as NLRC5, RFX-5, -ANK and -AP, IRF1, NF-Y, and CIITA.

[0125] In certain embodiments, HLA expression is disrupted.In some aspects, HLA expression is disrupted by targeting individual HLA (e.g., knocking out the expression of HLA-A, HLA-B and / or HLA-C), targeting the transcription regulator of HLA expression (e.g., knocking out the expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C and / or IRF-1), blocking the surface transport of MHC class I molecules (e.g., knocking out the expression of B2M and / or TAP1), and / or targeting HLA-Razor.

[0126] In certain embodiments, the stem cells disclosed herein are characterized as being hypoimmunogenic because they do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B, and / or HLA-C) corresponding to MHC-I and / or MHC-II. For example, in certain embodiments, the stem cells disclosed herein are modified such that the stem cells or differentiated stem cells prepared therefrom do not express or exhibit reduced expression of one or more of the following MHC-I molecules: HLA-A, HLA-B, and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C can be "knocked out" of the cell. Cells with knocked out HLA-A, HLA-B, and / or HLA-C genes can exhibit reduced or eliminated expression of each knocked out gene. See Figures 13A-J.

[0127] In some embodiments, the present disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof, comprising a genome in which the HLA-A gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. The contiguous stretch of genomic DNA can be deleted by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 2-1418.

[0128] In certain aspects, the disclosure provides a method for altering a target HLA-A sequence in a cell, comprising contacting the HLA-A sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid or at least one pair of ribonucleic acids, where the ribonucleic acids direct the Cas protein to and hybridize to a target motif in the target HLA-A polynucleotide sequence, where the target HLA-A polynucleotide sequence is cleaved, and where the at least one ribonucleic acid or the at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 2-1418.

[0129] In some embodiments, the present disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof, comprising a genome in which the HLA-B gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population. The contiguous stretch of genomic DNA can be deleted by contacting the cell or population with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 1419-3277.

[0130] In certain aspects, the disclosure provides a method for altering a target HLA-B sequence in a cell, comprising contacting the HLA-B sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid or at least one pair of ribonucleic acids, where the ribonucleic acids direct the Cas protein to and hybridize to a target motif in the target HLA-B polynucleotide sequence, where the target HLA-B polynucleotide sequence is cleaved, and where the at least one ribonucleic acid or the at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 1419-3277.

[0131] In some embodiments, the present disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof, comprising a genome in which the HLA-C gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population. The contiguous stretch of genomic DNA can be deleted by contacting the cell or population with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 3278-5183.

[0132] In certain aspects, the disclosure provides a method for altering a target HLA-C sequence in a cell, comprising contacting the HLA-C sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid or at least one pair of ribonucleic acids, where the ribonucleic acids direct the Cas protein to and hybridize to a target motif in the target HLA-C polynucleotide sequence, where the target HLA-C polynucleotide sequence is cleaved, and where the at least one ribonucleic acid or the at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3278-5183.

[0133] In certain embodiments, the expression of MHC-I or MHC-II is modulated by targeting and deleting a continuous stretch of genomic DNA, thereby reducing or eliminating the expression of target gene, such as NLRC5. As used herein, the term "modulate" is used in accordance with its use in the art (i.e., to cause or promote a qualitative or quantitative change, alteration or modification of a process, pathway or phenomenon of interest). Without being limited thereto, such change can be an increase, decrease or change in the relative strength or activity of different components or branches of a process, pathway or phenomenon. In certain aspects, the target gene is NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C or IRF-1.

[0134] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) the expression of MHC-II genes by targeting and modulating (e.g., reducing or eliminating) class II transcription activator (CIITA) expression. In some embodiments, the modulation is performed using a CRISPR / Cas system. CIITA is a member of the NLR or nucleotide binding domain (NBD) leucine-rich repeat (LRR) family of proteins, which regulates the transcription of MHC-II by associating with the MHC enhanceosome. In B cells and dendritic cells, the expression of CIITA is induced depending on the developmental stage and is inducible by IFN-γ in most cell types. Apart from CIITA, NLR proteins are localized in the cytoplasm and contribute to the innate immune response by recognizing microbial products and exogenous danger signals, resulting in inflammation and / or cell death.

[0135] In some aspects, the disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof comprising a genome in which the class II transcriptional activator (CIITA) gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof.

[0136] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 5184-36352.

[0137] The present invention contemplates genome editing of human cells to cleave CIITA gene sequences, and genome editing of such cells to modify one or more additional target polynucleotide sequences (e.g., NLRC5 and / or B2M).It should be recognized that cleavage of CIITA genome sequences using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of target CIITA genome sequences, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0138] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the CIITA gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class II molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352.

[0139] In some aspects, the present invention provides a method for altering a target CIITA polynucleotide sequence in a cell, comprising contacting the CIITA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct the Cas protein to and hybridize with a target motif in the target CIITA polynucleotide sequence, wherein the target CIITA polynucleotide sequence is cleaved, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 5184-36352.

[0140] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the CIITA gene has been edited to reduce or eliminate CIITA surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352.

[0141] In some aspects, the present invention provides a method for altering a target CIITA polynucleotide sequence in a cell, comprising contacting the CIITA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, wherein the ribonucleic acid directs and hybridizes the Cas protein to a target motif in the target CIITA polynucleotide sequence, wherein the target CIITA polynucleotide sequence is cleaved, and wherein the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 5184-36352.

[0142] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) the expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) the expression of NLR family CARD domain containing 5 / NOD27 / CLR16.1 (NLRC5). In some embodiments, the modulation is performed using a CRISPR / Cas system. NLRC5 is a key regulator of MHC-I mediated immune responses, and like CIITA, NLRC5 is highly inducible by IFN-γ and can translocate into the nucleus. NLRC5 activates the promoter of MHC-I genes and induces the transcription of related genes involved in MHC-I and MHC-I antigen presentation.

[0143] In some aspects, the disclosure provides a stem cell (e.g., a hypoimmunogenic stem cell) or population thereof comprising a genome in which the NLRC5 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0144] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 36353-81239.

[0145] The present invention contemplates genome editing of human cells to cleave the NLRC5 gene sequence, and editing the genome of such cells to modify one or more additional target polynucleotide sequences (e.g., CIITA and / or B2M). It should be recognized that cleavage of the NLRC5 genomic sequence using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of the target NLRC5 genomic sequence, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0146] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the NLRC5 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239.

[0147] In some aspects, the invention provides methods for altering a target NLRC5 polynucleotide sequence in a cell, comprising contacting the NLRC5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target NLRC5 polynucleotide sequence, where the target NLRC5 polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 36,353-81,239.

[0148] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the NLRC5 gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239.

[0149] In some aspects, the present invention provides a method for altering a target NLRC5 polynucleotide sequence in a cell, comprising contacting the NLRC5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target NLRC5 polynucleotide sequence, where the target NLRC5 polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 36353-81239.

[0150] In certain embodiments, the invention disclosed herein provides an accessory chain B2 By targeting and modulating (e.g., reducing or eliminating) the expression of M, the expression of MHC-I genes is modulated (e.g., reduced or eliminated). In some embodiments, the modulation is performed using the CRISPR / Cas system. By modulating (e.g., reducing or eliminating) the expression of B2M, the surface transport of MHC-I molecules is blocked, making the cells less immunogenic.

[0151] In some aspects, the present disclosure provides a stem cell (e.g., a hypoimmunogenic stem cell) or population thereof comprising a genome in which the beta2-microglobulin (B2M) gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0152] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 81240-85644.

[0153] The present invention contemplates genome editing of human cells to cleave the B2M gene sequence, and editing the genome of such cells to alter one or more additional target polynucleotide sequences (e.g., NLRC5 and / or CIITA). It should be recognized that cleavage of the B2M genomic sequence using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of the targeted B2M genomic sequence, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0154] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the B2M gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644.

[0155] In some aspects, the invention provides methods for altering a target B2M polynucleotide sequence in a cell, comprising contacting the B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target B2M polynucleotide sequence, where the target B2M polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 81240-85644.

[0156] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the B2M gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644.

[0157] In some aspects, the invention provides a method for altering a target B2M polynucleotide sequence in a cell, comprising contacting the B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target B2M polynucleotide sequence, where the target B2M polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 81240-85644.

[0158] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of one or more RFX5. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0159] In some aspects, the disclosure provides a stem cell (e.g., a hypoimmunogenic stem cell) or population thereof comprising a genome in which the RFX5 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0160] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 85645-90115.

[0161] The present invention contemplates genome editing of human cells to cleave RFX5 gene sequences, and editing the genome of such cells to alter one or more additional target polynucleotide sequences. It should be recognized that cleavage of the RFX5 genomic sequence using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of the targeted RFX5 genomic sequence, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0162] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the RFX5 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA has been deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 85645-90115.

[0163] In some embodiments, the invention provides methods for altering a target RFX5 polynucleotide sequence in a cell, comprising contacting the RFX5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target RFX5 polynucleotide sequence, where the target RFX5 polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 85645-90115.

[0164] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the RFX5 gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 85645-90115.

[0165] In some embodiments, the invention provides methods for altering a target RFX5 polynucleotide sequence in a cell, comprising contacting the RFX5 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target RFX5 polynucleotide sequence, where the target RFX5 polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 85645-90115.

[0166] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of one or more RFXAs. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0167] In some aspects, the disclosure provides a stem cell (e.g., a hypoimmunogenic stem cell) or population thereof comprising a genome in which the RFXAP gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0168] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 90116-95635.

[0169] The present invention contemplates genome editing of human cells to cleave the RFXAP gene sequence, and editing the genome of such cells to alter one or more additional target polynucleotide sequences. It should be recognized that cleavage of the RFXAP genomic sequence using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of the targeted RFXAP genomic sequence, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0170] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the RFXAP gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 90116-95635.

[0171] In some aspects, the invention provides methods for altering a target RFXAP polynucleotide sequence in a cell, comprising contacting the RFXAP polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target RFXAP polynucleotide sequence, where the target RFXAP polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 90116-95635.

[0172] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the RFXAP gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 90116-95635.

[0173] In some aspects, the invention provides methods for altering a target RFXAP polynucleotide sequence in a cell, comprising contacting the RFXAP polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target RFXAP polynucleotide sequence, where the target RFXAP polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 90116-95635.

[0174] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of one or more RFXANKs. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0175] In some aspects, the disclosure provides a stem cell (e.g., a hypoimmunogenic stem cell) or population thereof comprising a genome in which the RFXANK gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0176] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 95636-102318.

[0177] The present invention contemplates genome editing of human cells to cleave the RFXANK gene sequence, and editing the genome of such cells to modify one or more additional target polynucleotide sequences. It should be recognized that cleavage of the RFXANK genome sequence using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of the target RFXANK genome sequence, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0178] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the RFXAP gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 95636-102318.

[0179] In some aspects, the invention provides methods for altering a target RFXANK polynucleotide sequence in a cell, comprising contacting the RFXANK polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids target and hybridize the Cas protein to a target motif in the target RFXANK polynucleotide sequence, wherein the target RFXANK polynucleotide sequence is cleaved, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 95636-102318.

[0180] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the RFXANK gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 95635-102318.

[0181] In some aspects, the present invention provides a method for altering a target RFXANK polynucleotide sequence in a cell, comprising contacting the RFXANK polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target RFXANK polynucleotide sequence, where the target RFXANK polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 95636-102318.

[0182] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) the expression of MHC-I genes by targeting and modulating (e.g., reduces or eliminates) the expression of one or more NYF-A. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0183] In some aspects, the disclosure provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a genome in which the NFY-A gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof.

[0184] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 102319-121796.

[0185] The present invention contemplates genome editing of human cells to cut NFY-A gene sequence, and genome editing of such cells to modify one or more additional target polynucleotide sequences.It should be recognized that the cutting of NFY-A genome sequence using one or more gRNA or gRNA pairs and Cas protein described herein can result in partial or complete deletion of target NFY-A genome sequence, depending on the number of gRNA or gRNA pairs selected and their targets.

[0186] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the NFY-A gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 102319-121796.

[0187] In some aspects, the invention provides methods for altering a target NFY-A polynucleotide sequence in a cell, comprising contacting the NFY-A polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target NFY-A polynucleotide sequence, where the target NFY-A polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 102319-121796.

[0188] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the NFY-A gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 102319-121796.

[0189] In some aspects, the invention provides methods for altering a target NFY-A polynucleotide sequence in a cell, comprising contacting the NFY-A polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target NFY-A polynucleotide sequence, where the target NFY-A polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 102319-121796.

[0190] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of one or more NFY-B. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0191] In some aspects, the present disclosure provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a genome in which the NFY-B gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof.

[0192] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 121797-135112.

[0193] The present invention contemplates genome editing of human cells to cut NFY-B gene sequence, and genome editing of such cells to modify one or more additional target polynucleotide sequences.It should be recognized that the cutting of NFY-B genome sequence using one or more gRNA or gRNA pairs and Cas protein described herein can result in partial or complete deletion of target NFY-B genome sequence, depending on the number of gRNA or gRNA pairs selected and their targets.

[0194] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the NFY-B gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 121797-135112.

[0195] In some aspects, the invention provides methods for altering a target NFY-B polynucleotide sequence in a cell, comprising contacting the NFY-B polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target NFY-B polynucleotide sequence, where the target NFY-B polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 121797-135112.

[0196] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the NFY-B gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 121797-135112.

[0197] In some aspects, the invention provides methods for altering a target NFY-B polynucleotide sequence in a cell, comprising contacting the NFY-B polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target NFY-B polynucleotide sequence, where the target NFY-B polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 121797-135112.

[0198] In certain embodiments, the invention disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of one or more NFY-C. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0199] In some aspects, the disclosure provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a genome in which the NFY-C gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof.

[0200] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 135113-176601.

[0201] The present invention contemplates genome editing of human cells to cut NFY-C gene sequence, and genome editing of such cells to modify one or more additional target polynucleotide sequences.It should be recognized that the cutting of NFY-C genome sequence using one or more gRNA or gRNA pairs and Cas protein described herein can result in partial or complete deletion of target NFY-C genome sequence, depending on the number of gRNA or gRNA pairs selected and their targets.

[0202] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the NFY-C gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 135113-176601.

[0203] In some aspects, the invention provides methods for altering a target NFY-C polynucleotide sequence in a cell, comprising contacting the NFY-C polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target NFY-C polynucleotide sequence, where the target NFY-C polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 135113-176601.

[0204] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the NFY-C gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 135113-176601.

[0205] In some aspects, the invention provides methods for altering a target NFY-C polynucleotide sequence in a cell, comprising contacting the NFY-C polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target NFY-C polynucleotide sequence, where the target NFY-C polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 135113-176601.

[0206] In certain embodiments, the inventions disclosed herein modulate (e.g., reduce or eliminate) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of one or more IRF-1. In some embodiments, modulation is performed using a CRISPR / Cas system.

[0207] In some aspects, the disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof comprising a genome in which the IRF-1 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0208] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 176602-182813.

[0209] The present invention contemplates genome editing of human cells to cleave IRF-1 gene sequences, and genome editing of such cells to modify one or more additional target polynucleotide sequences.It should be recognized that cleavage of IRF-1 genome sequences using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of target IRF-1 genome sequences, depending on the number of gRNAs or gRNA pairs selected and their targets.

[0210] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the IRF-1 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 176602-182813.

[0211] In some aspects, the invention provides methods for altering a target IRF-1 polynucleotide sequence in a cell, comprising contacting the IRF-1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target IRF-1 polynucleotide sequence, where the target IRF-1 polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 176602-182813.

[0212] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a modified genome comprising a genomic modification in which the IRF-1 gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 176602-182813.

[0213] In some aspects, the invention provides methods for altering a target IRF-1 polynucleotide sequence in a cell, comprising contacting the IRF-1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target IRF-1 polynucleotide sequence, where the target IRF-1 polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 176602-182813.

[0214] In some aspects, the disclosure provides a stem cell (e.g., a hypoimmunogenic stem cell) or population thereof comprising a genome in which the TAP1 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof.

[0215] The contiguous stretch of genomic DNA can be deleted by contacting a cell or a population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 182814-188371.

[0216] The present invention contemplates genome editing of human cells to cut TAP1 gene sequence, and genome editing of such cells to modify one or more additional target polynucleotide sequences.It should be recognized that the cutting of TAP1 genome sequence using one or more gRNA or gRNA pairs and Cas protein described herein can result in partial or complete deletion of target TAP1 genome sequence, depending on the number of gRNA or gRNA pairs selected and their targets.

[0217] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, each cell comprising a modified genome comprising a genomic modification in which the TAP1 gene has been edited to delete a contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA is deleted by contacting the cell with a pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 182814-188371.

[0218] In some aspects, the invention provides methods for altering a target TAP1 polynucleotide sequence in a cell, comprising contacting the TAP1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and one to two ribonucleic acids, where the ribonucleic acids target and hybridize the Cas protein to a target motif in the target TAP1 polynucleotide sequence, where the target TAP1 polynucleotide sequence is cleaved, and where at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 182814-188371.

[0219] In some aspects, the present invention provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof that comprise a modified genome comprising a genomic modification in which the TAP1 gene has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 182814-188371.

[0220] In some aspects, the invention provides methods for altering a target TAP1 polynucleotide sequence in a cell, comprising contacting the TAP1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats associated (Cas) protein and at least one ribonucleic acid, where the ribonucleic acid targets and hybridizes the Cas protein to a target motif in the target TAP1 polynucleotide sequence, where the target TAP1 polynucleotide sequence is cleaved, and where the at least one ribonucleic acid is selected from the group consisting of SEQ ID NOs: 182814-188371.

[0221] In certain embodiments, the gRNA that allows simultaneous deletion of all MHC class I alleles by targeting the conserved region in HLA gene is identified as HLA Razor.In some aspects, gRNA is part of CRISPR system.In alternative aspects, gRNA is part of TALEN system.In one aspect, the HLA Razor that targets the identified conserved region in HLA is represented in Figure 21A.In other aspects, multiple HLA Razors that target the identified conserved region are utilized.It is generally understood that any guide that targets the conserved region in HLA can act as HLA Razor. Knock-in

[0222] In certain embodiments, tolerogenic factors can be inserted or reinserted into genome-edited stem cell lines to create immune-privileged universal donor stem cell lines. In certain embodiments, the universal stem cells disclosed herein are further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitor and IL-35.

[0223] The present inventors used genome editing systems, such as the CRISPR / Cas system, to facilitate the insertion of tolerogenic factors (e.g., the tolerogenic factors shown in Table 2 below) into safe harbor loci (e.g., the AAVS1 locus) to actively inhibit immune rejection. As seen in Figures 11A-11C, the present inventors successfully expressed tolerogenic factors (e.g., PD-L1 and HLA-G) from the safe harbor loci.

[0224] [Table 2]

[0225] In some aspects, the disclosure provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof, comprising a genome in which the stem cell genome has been modified to express HLA-G. In some aspects, the disclosure provides methods for modifying a stem cell genome to express HLA-G. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-G into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 188372-189858.

[0226] In some aspects, the disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof, comprising a genome in which the stem cell genome has been modified to express HLA-C. In some aspects, the disclosure provides a method for modifying a stem cell genome to express HLA-C. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-C into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 3278-5183.

[0227] In some aspects, the disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof, comprising a genome in which the stem cell genome has been modified to express HLA-E. In some aspects, the disclosure provides a method for modifying a stem cell genome to express HLA-E. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-E into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 189859-193183.

[0228] In some aspects, the disclosure provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a genome in which the stem cell genome has been modified to express PD-L1. In some aspects, the disclosure provides methods for modifying a stem cell genome to express PD-L1. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of PD-L1 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 193184-200783.

[0229] In some aspects, the disclosure provides stem cells (e.g., hypoimmunogenic stem cells) or populations thereof comprising a genome in which the stem cell genome has been modified to express CD-47. In some aspects, the disclosure provides methods for modifying a stem cell genome to express CD-47. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of CD-47 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 200784-231885.

[0230] In some aspects, the disclosure provides a stem cell (e.g., hypoimmunogenic stem cell) or population thereof, comprising a genome in which the stem cell genome has been modified to express HLA-F. In some aspects, the disclosure provides a method for modifying a stem cell genome to express HLA-F. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be utilized to facilitate insertion of HLA-F into a stem cell line. In certain embodiments, the at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs: 688808-399754. Other targeted modifications

[0231] In some embodiments, additional targets may be modified and / or deleted in universal cells, such as universal T cells, to improve their function and / or tailor them to specific therapeutic approaches.

[0232] In some embodiments, genes encoding costimulatory molecules / receptors involved in cytotoxic T cells can be deleted by genome editing (Table 3). Deletion of costimulatory molecules / receptors can be performed to prevent autoimmunity. In other embodiments, genes encoding co-inhibitory molecules / receptors can be deleted by genome editing (Table 4). Deletion of co-inhibitory molecules / receptors can be performed to prevent T cell inhibition by cancer cells, which can be useful in T cell-based cancer immunotherapy.

[0233] [Table 3]

[0234] [Table 4]

[0235] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the OX40 gene is edited to modify (e.g., delete) a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target OX40 sequence in a cell. The continuous stretch of genomic DNA can be modified (possibly deleted) by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 231886-234210.

[0236] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the GITR gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target GITR sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 234211-236445.

[0237] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the 4-1BB gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target 4-1BB sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 234211-236445.

[0238] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD28 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD28 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 252807-274181.

[0239] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the B7-1 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target B7-1 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 274182-295529.

[0240] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the B7-2 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target B7-2 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 295530-324177.

[0241] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the ICOS gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target ICOS sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 324178-339974.

[0242] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD27 gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD27 sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 339975-344266.

[0243] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the HVEM gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target HVEM sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 344267-350722.

[0244] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which a SLAM gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target SLAM sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 350723-353590.

[0245] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the CD226 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD226 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 353591-416840.

[0246] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the PD1 gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target PD1 sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of sequences numbered 196-531 and 4047-9101 in U.S. Patent Application No. 15 / 083,021, which is incorporated herein by reference.

[0247] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CTLA4 gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CTLA4 sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of the sequences numbered 1-195 and 797-4046 in U.S. Patent Application No. 15 / 083,021, which is incorporated herein by reference.

[0248] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the LAG3 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target LAG3 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 416841-421195.

[0249] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the TIGIT gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target TIGIT sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 421196-432039.

[0250] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the TIM3 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target TIM3 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 432040-447610.

[0251] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD160 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD160 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 447611-459294.

[0252] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the BTLA gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target BTLA sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 459295-482454.

[0253] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD244 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD244 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 482455-504169.

[0254] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD244 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD244 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 482455-504169.

[0255] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD30 gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD30 sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 699755-731993.

[0256] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the TLT gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target TLT sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 731994-739957.

[0257] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the VISTA gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target VISTA sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 739958-757515.

[0258] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the B7-H3 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target B7-H3 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 757516-777888.

[0259] In some embodiments, the present disclosure provides a stem cell or population thereof comprising a genome in which the PD-L2 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for altering a target PD-L2 sequence in a cell. The contiguous stretch of genomic DNA may be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 777889-817976.

[0260] In some embodiments, lymphocyte adhesion is blocked to prevent autoimmunity. In certain aspects, target genes can be edited by genome editing to block lymphocyte adhesion (Table 5).

[0261] [Table 5]

[0262] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the LFA-1 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target LFA-1 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 504170-526670.

[0263] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD2 gene is edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD2 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 526671-536704.

[0264] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the CD58 gene has been edited to modify a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target CD58 sequence in a cell. The contiguous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 536705-570967.

[0265] In some embodiments, the gene encoding the T cell receptor can be deleted by genome editing. The deletion of the T cell receptor gene can be performed to prevent autoimmune attacks in T cell therapy. In some aspects, the gene encoding the T cell receptor is the T cell receptor alpha locus (TCRA) or its homolog, ortholog, or variant (also known as Gene ID: 5133, IMD7, TCRD, TRA@, TRAC, and referred to herein as TCRa, TCRA, TCR alpha, etc.). In some aspects, the gene encoding the T cell receptor is the T cell receptor alpha locus (TCRB) or its homolog, ortholog, or variant (also known as Gene ID: 6957, TCRB; TRB@, and referred to herein as TCRb, TCRB, TCR beta, etc.). In some embodiments, the T cell receptor genes are modified by genome editing as described in U.S. Patent Application No. 15 / 083,021 and PCT Application No. PCT / US2016 / 024554, both of which are incorporated herein by reference.

[0266] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the TRAC gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target TRAC sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of sequences numbered 532-609 and 9102-9797 in U.S. Patent Application No. 15 / 083,021, which is incorporated herein by reference.

[0267] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the TRBC gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target TRBC sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of sequences numbered 610-765 and 9798-10573 in U.S. Patent Application No. 15 / 083,021, which is incorporated herein by reference.

[0268] In some embodiments, genes involved in regulatory T cell (Treg) function can be deleted by genome editing (Table 6). Deletion of genes involved in regulatory T cell (Treg) function can be performed to break tolerance in T cell therapy.

[0269] [Table 6]

[0270] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population comprising a genome in which the FOXP3 gene has been edited to modify (e.g., delete) a contiguous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target FOXP3 sequence in a cell. The contiguous stretch of genomic DNA can be modified (e.g., deleted) by contacting a cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 570968-584068.

[0271] In some embodiments, the present disclosure provides a stem cell or population thereof, the stem cell or population thereof comprising a genome in which the HELIOS gene is edited to modify a continuous stretch of genomic DNA. In certain embodiments, the present disclosure provides a method for modifying a target HELIOS sequence in a cell. The continuous stretch of genomic DNA can be modified by contacting the cell or population thereof with a Cas protein or a nucleic acid encoding a Cas protein, and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 683033-688807.

[0272] The multiplexing capabilities of the CRISPR / Cas system also allow for the creation of disease-specific universal donor cell lines with one or more genomic alterations that improve their applicability for treating specific diseases or conditions. A list of potential diseases that can be addressed can be found in Table 7.

[0273] [Table 7]

[0274] It is to be understood that the inventions disclosed herein are not limited in their application to the details shown or illustrated in this description. The invention encompasses other embodiments and can be practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0275] Although specific compositions, methods and assays of the present invention have been described in detail in accordance with particular embodiments, the following examples are illustrative only and are not intended to be limiting of the methods and compositions of the present invention.

[0276] The articles "a" and "an" as used herein should be understood to include plural referents unless the specification and claims clearly indicate to the contrary. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless otherwise indicated or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process. Moreover, it should be understood that the present invention encompasses all variations, combinations, and permutations, where one or more limits, elements, clauses, descriptive terms, etc. from one or more of the claims listed herein may be introduced into another claim according to the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it is obvious to one skilled in the art that a contradiction or inconsistency would result. When elements are presented as a list, e.g., in a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed, and any element may be removed from the group. In general, when the invention, or aspects of the invention, are referred to as comprising certain elements, features, etc., it should be understood that the particular embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements, features, etc. For the sake of brevity, these embodiments have not in every instance been specifically described in so many words herein. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is listed in the specification.Publications and other reference materials mentioned herein to describe the background of the invention and to provide further details regarding its practice are incorporated herein by reference. EXAMPLES

[0277] cell line We targeted different genes in different cell lines. The different cell lines used include HuES8, HuES9, BJ-RiPSC, Thp1, Jurkat, primary T cells and HEK293T cells (Figure 27). HuES8 and HuES9 are human ES cell lines. BJ-RiPSC is an iPSC line. Knockout of HLA-A, HLA-B and HLA-C

[0278] Knockout targeting of HLA was investigated. First, HLA-B and HLA-C knockout strategies were investigated. Two short guide RNAs (sgRNAs) were designed upstream of the HLA-B locus and downstream of HLA-C, allowing excision of the HLA-B and HLA-C genes (Figure 14A). sgRNA#1 and sgRNA#2 target the HLA-B upstream region, and sgRNA#3 and sgRNA#4 target the HLA-C downstream region.

[0279] PCR screening was performed to confirm that clone 1D was a homozygous knockout clone. A schematic showing the PCR validation strategy for successful deletion of HLA-B and HLA-C is shown in Figure 13B. Two pairs of wild-type (WT) primers flanking each cleavage site were designed with predicted amplicon sizes of 545 bp and 472 bp. Clone 1D was identified as a homozygous knockout clone by the presence of a PCR band of approximately 680 bp generated with the KO primers and the absence of a band when two different sets of WT primers were used (Figure 14B). In addition, genomic DNA was isolated from the targeted HuES8 clones shown. The results of sequencing the PCR products of clone 1D demonstrated the successful deletion of HLA-B and HLA-C genes in HuES9 (Figure 14C).

[0280] Furthermore, RT-PCR using HLA-B and HLA-C specific primers demonstrated the elimination of HLA-B and HLA-C mRNA expression in clone 1D (Figure 14D). GAPDH, TOP1 and HPRT1 were used as internal controls. RT-PCR products of WT and clone 1D amplified with HLA-B primers were sequenced and identified as HLA-B and HLA-A mRNA, respectively, using BLAST (Figure 14E). These results demonstrated that in the absence of HLA-B gene, HLA-B specific primers amplify HLA-A mRNA in HuES8 clone 1D. HuES8 clone 1D shows a normal karyotype as assessed by NanoString nCounter set (Figure 14F).

[0281] A schematic demonstrating the HLA-A knockout strategy using the dual sgRNA approach is provided in Figure 14G. The schematic shows the location of two sgRNAs (#5 and #6) designed to bind upstream and downstream of HLA-A. The on-target cleavage efficiency of sgRNAs #5 and #6 was determined in 293T cells using TIDE (Figure 14H).

[0282] PCR screening confirmed that clone 4E is a heterozygous HLA-A knockout clone (Figure 14I). The PCR screening strategy confirmed the deletion of HLA-A in HuES8. KO primers were designed with one primer annealing upstream and one primer annealing downstream of the cleavage site. With HLA-A deletion, the resulting amplicon was observed as 220 bp on a 2% agarose gel. Two pairs of WT primers were designed flanking each cleavage site with predicted amplicon sizes of 589 bp and 571 bp. Clone 4E was identified as a heterozygous clone by the presence of bands generated with the KO and WT primers amplified from genomic DNA (Figure 14I). Sequencing of the PCR products amplified from genomic DNA of clone 4E using the KO primers demonstrated the successful deletion of HLA-A in HuES8 (Figure 14J). Knockout of transcriptional regulators

[0283] As shown in Figures 3A-3B, in certain embodiments, the invention disclosed herein targets transcriptional regulators of antigen presentation (e.g., CIITA and / or NLRC5). The inventors have discovered that targeting and / or modulating the expression of such transcriptional regulators of antigen presentation effectively modulates (e.g., reduces or eliminates) the expression of three classical MHC-Ia molecules (HLA-A, HLA-B and HLA-C), thereby providing a means to generate hypoimmunogenic pluripotent stem cell lines useful for cell replacement therapy. As seen in Figure 4, the inventors have demonstrated that such genome-edited cells are characterized by reduced MHC-I expression compared to WT HuES9 cells, as assessed by FACS. Reduction or elimination of MHC-I surface expression was observed in stem cells in which the expression of NLRC5, CIITA and B2M was modulated using the CRISPR / Cas system. In particular, Figure 4 reveals that low basal MHC-I expression in stem cells can be increased by IFN-γ, and IFN-γ-treated NLRC5- / - In stem cells, a reduction of approximately 50% in MHC-I surface expression was observed, as well as a reduction in B2M - / - They demonstrate that a complete loss of MHC-I surface expression was observed in stem cells.

[0284] Similar results were observed for MHC-II expression after modulating the expression of CIITA. - / - The stem cell line was differentiated into macrophages, which are antigen-presenting cells. As shown in Figure 5C, CIITA-deficient stem cell-derived macrophages lacked MHC-II expression, clearly displaying the expected phenotype. Notably, by targeting the first coding exon of the CIITA gene (Figures 10A-10B), the above results demonstrate that MHC-II expression can be efficiently abrogated.

[0285] We next evaluated the functional consequences of reducing HLA expression in genome-edited cells in a humanized mouse model prepared by reconstituting an immunodeficient mouse model with the human immune system (Figure 6). As shown in Table 1 below, a similar number of teratomas formed in all cases, regardless of genotype and total colonies, had to be sacrificed. Similarly, no differences in the failure rate of teratoma formation were observed. We scrutinized the overall morphology of the teratoma samples more closely and identified distinct phenotypes that correlated with the level of MHC-I expression. MHC-I on the surface was reduced in NLRC5 and absent in B2M. Figure 7 shows the quantification of the teratoma types obtained. Furthermore, Figures 8A-C demonstrate CD8+ T-cell proliferation in wild-type cells, suggesting immune rejection of wild-type teratomas and improved engraftment of genome-edited cells in the humanized mouse model.

[0286] [Table 1]

[0287] Thus, the above results demonstrate successful targeting of NLRC5, CIITA and B2M in HuES9 and BJ RiPSCs, and further demonstrate that the genome-edited cells can differentiate into a variety of different cell types characterized by reduced HLA expression.

[0288] We have used both TALEN and CRISPR / Cas systems to facilitate targeting of transcriptional regulators of HLA expression (i.e., MHC enhanceosomes). TALEN-induced CIITA and NLRC5 mutations in BJ-RIPSC and HuES9 are shown in Figure 15A-D. In addition, CRISPR can also be used to target NLRC5 and CIITA to achieve reduced MHC class I expression and complete loss of MHC class II expression, respectively.

[0289] In Thp1 cells, CRISPR was used to target NLRC5, demonstrating that NLRC5- / -Thp1 cells had reduced MHC-I expression (Figure 16A). Examples of CRISPR and / or TALEN systems targeting NLRC5, CIITA and B2M are provided in Figure 16B. After targeting with NLRC5 CRISPR or B2M CRISPR, reduced MHC class I expression was shown in HuES9. For example, IFNγ-treated NLRC5- / - cells showed approximately 50% reduction, and B2M- / - cells showed complete loss of MHC-I surface expression (Figure 16C).

[0290] A two-component (e.g., dual vector) system was used to perform lentiviral transduction of Thp-1 (Figure 16D). The two-component system included Lenti-Cas9-Blasticidin and Lenti-Guide-puro. An example of different CRISPRs used for lentiviral transduction of Thp-1 is provided in Figure 16E. Thp-1 was transduced with lentiviruses encoding NLRC5 and CIITA. B2M CRISPR was used as a positive control. All cells were stimulated ON with 50U IFNγ to enhance HLA expression (HLA-A2 was 1:200 and HLA-DR was 1:100). CIITA and NLRC5 were demonstrated to act independently on MHC-II and MHC-I, respectively (Figure 16F). Ten days after CRISPR transduction, Thp1 cells were stimulated all ON with 50 U IFNγ (HLA-A2 at 1:200 and HLA-DR at 1:100), demonstrating that targeting of IRF1 resulted in loss of MHC-II expression (Figure 16G).

[0291] We further investigated the targeting of IRF1 and the resulting reduction of MHC class I expression in human pluripotent stem cells (HuES9) and Thp-1 cells. A schematic diagram demonstrating the dual CRISPR strategy for targeting the IRF1 locus is provided in Figure 17A. Different IRF1 guide combinations were tested (Figure 17B), and the "dual guide strategy" for the targeted deletion of IRF1 was shown (Figure 17C). After applying the dual guide strategy for the targeted deletion of IRF1, sequence confirmation of IRF1 CRISPR-induced deletion was provided (Figure 17D), and then HuES9 cells targeted with IRF1 were screened (Figure 17E). The presence of PCR bands suggests the successful targeting using the dual CRISPR strategy.

[0292] We then reconfirmed (Figure 17F) and genotyped (Figure 17G) the IRF1 clones. Sequencing confirmed IRF1 CRISPR-induced deletion in clone 12 (Figure 17H), clone 17 (Figure 17I) and clone 21 (Figure 17J). After 48 hours of IFNγ treatment, IRF1- / - HuES9 clones showed impaired MHC class I induction (Figure 17K).

[0293] We also investigated the targeting of additional enhanceosome components. Although we investigated the effect of targeting additional enhanceosome components on MHC-I expression in 293T cells, we expect that targeting these additional enhanceosome components will also affect MHC-II levels in cells that actually express them (e.g., Thp1 cells or other APCs). For example, we used a dual-guide strategy to investigate CRISPR targeting of RFX5 (Figure 18A), RFX-ANK (Figure 18C) and RFK-AP (Figure 18G) in 293T cells. Targeting results demonstrated reduced MHC class I expression of RFX5 (Figure 18B, Figure 18E-18F), RFK-ANK (Figure 18D, Figure 18E-F) and RFK-AP (Figure 18H). We also investigated CRISPR dual guide targeting of NFY-A (Figure 19A), NFY-B (Figure 19D) and NFY-C (Figure 19B) in 293T cells. Targeting results demonstrated a reduction in MHC class I expression of NFY-A (Figure 19C), NFY-B (Figure 19E) and NFY-C (Figure 19C). Blockade of MHC class I surface transport

[0294] In PCT Patent Application No. PCT / US2015 / 059621 (which is incorporated herein by reference and shown in Figures 32-40), we previously described successful targeting of B2M utilizing TALENs and / or CRISPR. It has further been demonstrated that surface expression of MHC class I molecules is reduced or eliminated, thereby blocking surface trafficking of MHC-I molecules. For example, loss of MHC class I surface expression in B2M- / - Jurkat Cas9 T cells was shown compared to MHC class I surface expression in Jurkat Cas9 T cells (Figure 31).

[0295] In addition, we have demonstrated that the surface transport of MHC class I molecules can be suppressed by disrupting the TAP1 gene, which is an ER-resident peptide transporter.For example, after 48 hours of treatment with IFNγ, TAP1 CRISPR expression reduces MHC-I surface expression in Jurkat cells (Figure 20A) and Jurkat T cells (Figure 20B).It has further been demonstrated that HLA surface expression can be eliminated in Jurkat (Cas9) T cells (Figure 20C and Figure 20D).Jurkat T cells were established from the peripheral blood of a 14-year-old boy with acute T cell leukemia. HLA Razor

[0296] We investigated CRISPR guide RNAs that allow simultaneous deletion of all MHC class I alleles by targeting conserved regions in HLA genes. We also identified TALEN pair guides that target the same conserved regions in MHC class I genes. Any guide that targets conserved regions can be identified or classified as HLA Razor. Targeting of conserved sequences found in all HLA by CRISPR or TALENS was demonstrated in Figure 21A. Two purple boxes indicate the binding sites of pan-HLA TALEN pairs. Blue arrows indicate the pan-HLA CRISPR pairs tested, and PAM is circled in blue. Expression of pan-HLA-TALENs in 293T and HuES9 cells 72 hours after transfection was shown in Figure 21B. As demonstrated in Figure 21C, HLA-Razor CRISPR attenuates MHC class I expression in 293T cells. Cas9-GFP was also co-transfected into 293T cells. Figure 21D provides a comparison of the activity of two different HLA Razors targeting two different conserved regions of HLA. PD-L1 and HLA-G knock-in

[0297] The present inventors used the CRISPR / Cas system to facilitate the insertion of tolerogenic factors (e.g., those shown in Table 2) into the AAVS1 locus to actively inhibit immune rejection. As shown in Figures 11A-11C, we successfully expressed tolerogenic factors (e.g., PD-L1 and HLA-G) from the AAVS1 locus.

[0298] A schematic of the PD-L1 and HLA-G knock-in strategy is shown in Figure 22A. Wild-type (WT) and knock-in (KI) primers for clone screening were designed. The amplicon with WT primers is predicted to be 488bp, and the amplicon with KI primers is predicted to be 403bp and 915bp. The design of the knock-in donor plasmid (Figure 22B) shows that the reading frames of PD-L1 and HLA-G are linked by T2A and their expression is driven by the CAGGS promoter. Puromycin was used as a drug resistance marker after the SA-2A gene trap element.

[0299] In 293T cells transfected with donor plasmid, the expression of PD-L1 and HLA-G was investigated by FACS analysis. Figure 22C shows ectopic PD-L1 and HLA-G expression in 293T cells. APC-conjugated PD-L1 antibody and FITC-conjugated HLA-G antibody were used. In addition, the expression of ectopic HLA-G in JEG-3 cells is shown in Figure 22D. The donor plasmid was transfected into HLA-G- / -JEG-3 cell line, and 48 hours after transfection, the ectopic HLA-G expression was investigated by FACS analysis. PE-conjugated HLA-G antibody (MEM / G9) was used to detect surface HLA-G surface expression.

[0300] PCR screening was performed to confirm that clone 1G was a PD-L1 / HLA-G heterozygous KI clone (Figure 22E). Clone 1G was identified as a heterozygous KI clone by the presence of a band amplified from HuES8 genomic DNA targeted with both WT and KI-specific primers. PD-L1 expression was verified in HuES8 KI clone 1G by FACS analysis using APC-conjugated PD-L1 antibody (Figure 22F). Knock-in of CD-47

[0301] In addition, a CD47 knock-in strategy was also investigated. Human CD47 was cloned into an expression plasmid driven by a CAG promoter, which also contained IRES-GFP. 293T cells already express high levels of CD47 (human "don't eat me" signal), which prevents phagocytosis of cells by macrophages. In both 293T cells and human pluripotent stem cells (HuES9), expression could be increased by overexpression of CD47, as detected by FACS using a CD47-specific antibody (Figure 23). Overexpression of CD47 supports the engraftment of stem cell-derived transplants by protecting cells from macrophage phagocytosis. Further targets to be modified in pluripotent donor cells

[0302] To tailor the universal donor cells to specific applications, additional targets can be modified in them.For example, to tailor the universal donor cells to use as universal CAR T cells, additional targets can be modified in them.In one example, we deleted TRAC and TRBC in HuES9 to disrupt TCR expression (Figure 24).Using dual guide RNA approach, we introduced deletion into TRAC locus and TRBC locus in HuES9 cells.TCRA wild type band is 249bp, and after deletion, it is 209bp.TCRB wild type band is 162bp, and after deletion, it is 140bp. We further targeted TCRA in HuES9 B2M- / -CIITA- / - to create a B2M- / -, CIITA- / - and TCR- / - triple knockout stem cell line, which upon differentiation into T cells lacks MHC-I and MHC-II and shows no TCR surface expression.

[0303] In another example, we targeted PD-L1 (Figure 25A). CRISPR targeting Cd274 / B7-H1 / PD-L1 was found to be very useful in breaking tolerance in cancer immunotherapy. PD-L1 knockout was demonstrated in multiple cell types, including JEG-3 (a choriocarcinoma cell line), and in two melanoma cell lines (501 and MalMe) (Figures 25B-25D). Screening of targeted B7-H1 colonies was performed and found to have a CRISPR cutting efficiency of 8.3% in JEG-3 cells (Figure 25B). In addition, reconfirmation of the 501 melanoma knockout clone was shown in Figure 25C. Reconfirmation of the MalMe melanoma knockout clone was shown in Figure 25D. Targeting co-inhibitor / co-stimulatory receptors or their ligands

[0304] We have utilized CRISPR to investigate multiple targets. An illustration of costimulatory / inhibitory molecules and their receptors on T cells is provided in Figure 26A. For each target investigated, the four CRISPRs shown were cloned and tested for on-target activity in 293T cells. The expected size of the PCR bands when cleavage of both CRISPRs occurs is shown below each individual gel image (Figures 26B-26P). Dual guide targeting was demonstrated in 293T cells for TIGIT (Figure 26B), TIM3 (Figure 26C), HVEM (Figure 26D), 2B4 / CD244 (Figure 26E), CD28 (Figure 26F), OX40 (Figure 26G), B71 (Figure 26H), CD226 (Figure 26I), CD2 (Figure 26J), LAG3 (Figure 26K), BTLA (Figure 26L), ICOS (Figure 26M), CD27 (Figure 26N), ST2 (Figure 26O) and GITR (Figure 26P).

[0305] CRISPR targeting of B7-H3 was investigated in JEG-3 cells (Figures 30A-30D). Figure 30B shows the screening of targeted B7-H3 colonies in JEG-3 cells, showing that the CRISPR cutting efficiency is 15 / 80, or about 18.7%. Confirmation of B7-H3 knockout by sequencing was performed (Figure 30C), and the loss of B7-H3 surface expression in targeted Jeg3 clones is provided in Figure 30D. Differentiation of modified embryonic stem cells

[0306] Modified embryonic stem cells can differentiate into a variety of different cell types with reduced or absent HLA expression (Figure 28A). Examples of such cell types include mesenchymal progenitor cells (MPCs), hypoimmunogenic cardiomyocytes, endothelial cells (ECs), macrophages, hepatocytes, beta cells (e.g., pancreatic beta cells) or neural progenitor cells (NPCs).

[0307] First, in Figure 28B, we demonstrated a reduction in MHC-I expression in NLRC5- / - human ES cells. Although low basal MHC-I expression was observed in stem cells, expression could be increased by IFNγ stimulation. In IFNγ-treated NLRC5- / - cells, it was reduced by about 50%.

[0308] We then investigated MHC-I expression in various differentiated cell types. For example, Figure 28C shows the reduction of MHC-I expression in NLRC5- / - human mesenchymal progenitor cells (MPCs). The graph included in Figure 28C demonstrates the difference in MHC-I expression in HuES9 cells and MPC cells. Figure 28D shows the reduction of MHC-I expression in stem cell-derived NLRC5- / - endothelial cells (ECs). Similar differentiation efficiency for ECs is shown in Figure 28E, and the loss of HLA expression in B2M- / -CIITA- / -ECs is shown in Figure 28F. Next, we provided the reduction of MHC class I expression in NLRC5- / - hepatocyte-like cells (HLCs) (Figure 28G). HLCs were generated from BJ-RIPSCs.

[0309] In Figure 28H, the CIITA mutation is shown to abrogate MHC class II expression in hESC-derived macrophages. In addition, neural progenitor cells (NPCs) are differentiated from embryonic stem cells (Figure 28I). B2M- / -CIITA- / -HuES9 cells were shown to form nestin+ neural rosettes as a result of differentiation (white arrows in the figure). Finally, we adapted the modified embryonic stem cells to spin culture for use in β-cell differentiation (Figure 28J). In vivo data

[0310] Finally, we created "low immunogenic" DKO cell lines (Figure 29A). The cell lines investigated were WT HuES9 cells, NLRC5- / -C2TA- / -HuES9 cells and B2M- / -C2TA- / -HuES9 cells. Different cell lines showed different levels of MHC-I and MHC-II expression (Figure 29A). For example, WT HuES9 cells showed MHC-I and MHC-II expression. NLRC5- / -C2TA- / -HuES9 cells showed reduced MHC-I expression and no MHC-II expression. B2M- / -C2TA- / -HuES9 cells showed no MHC-I and no MHC-II expression. These cell lines were then investigated in humanized mice, which showed improved engraftment of genome-edited stem cells in humanized mice (Figure 29B). The present invention provides, for example, the following items. (Item 1) A stem cell comprising modulated expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens as compared to wild-type stem cells. (Item 2) A stem cell comprising modulated expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens compared to a wild-type stem cell, wherein the hypoimmunogenic stem cell further comprises one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II deleted from at least one allele of the cell. (Item 3) The stem cell according to items 1 to 2, wherein the modulated expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises reduced expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens. (Item 4) The stem cell according to items 1 to 2, wherein the modulated expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises inhibition of expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens. (Item 5) The stem cell according to items 1 to 2, wherein the modulated expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises disrupting the expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens. (Item 6) The stem cell of item 1, wherein the modulated expression of the one or more MHC-I and MHC-II human leukocyte antigens comprises one or more genes encoding the one or more MHC-I and MHC-II human leukocyte antigens deleted from at least one allele of the cell. (Item 7) The stem cell according to item 6, wherein the transcriptional regulator of MHC-I or MHC-II is selected from the group consisting of NLRC5, CIITA and B2M. (Item 8) The stem cell of item 6, wherein the transcriptional regulator of MHC-I or MHC-II comprises NLRC5. (Item 9) The stem cell according to item 6, wherein the transcriptional regulator of MHC-I or MHC-II comprises CIITA. (Item 10) The stem cell according to item 6, wherein the transcriptional regulator of MHC-I or MHC-II comprises B2M. (Item 11) The stem cell of item 1, further comprising modulated expression of one or more tolerogenic factors compared to wild-type stem cells. (Item 12) 12. The stem cell of item 11, wherein the modulated expression of the tolerogenic factor comprises increased expression of the tolerogenic factor. (Item 13) 12. The stem cell of item 11, wherein the tolerogenic factor is inserted into a safe harbor locus of at least one allele of the cell. (Item 14) Item 15. The stem cell according to item 11, wherein the safe harbor locus comprises the AAVS1 locus. The stem cell of item 11, wherein the tolerogenic factor inhibits immune rejection. (Item 16) 12. The stem cell according to item 11, wherein the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitory factor and IL-35. (Item 17) 3. The stem cell according to items 1 to 2, which is an embryonic stem cell. (Item 18) 3. The stem cell according to items 1 to 2, which is a pluripotent stem cell. (Item 19) The stem cell according to items 1 to 2, which is low immunogenic. (Item 20) 3. The stem cell according to items 1 to 2, which is a human stem cell. (Item 21) 3. The stem cell according to items 1 to 2, which does not express one or more of NLRC5, CIITA and B2M. (Item 22) 3. The stem cell according to items 1 to 2, which does not express one or more of HLA-A, HLA-B and HLA-C. (Item 23) 3. The stem cell according to items 1 to 2, which has reduced expression of one or more of HLA-A, HLA-B and HLA-C compared to a wild-type stem cell. (Item 24) A stem cell comprising modulated expression of one or more tolerogenic factors as compared to wild-type human stem cells. (Item 25) 25. The stem cell of item 24, wherein the modulated expression of the tolerogenic factor comprises increased expression of the tolerogenic factor. (Item 26) 25. The stem cell of item 24, wherein the tolerogenic factor is inserted into a safe harbor locus of at least one allele of the cell. (Item 27) Item 28. The stem cell according to Item 26, wherein the safe harbor locus comprises the AAVS1 locus. 25. The stem cell of item 24, wherein the tolerogenic factor inhibits immune rejection. (Item 29) 25. The stem cell according to item 24, which is an embryonic stem cell. (Item 30) 25. The stem cell according to item 24, which is a pluripotent stem cell. (Item 31) 25. The stem cell according to item 24, which is low immunogenic. (Item 32) 25. The stem cell according to item 24, which is a human stem cell. (Item 33) 25. The stem cell according to item 24, wherein the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitory factor and IL-35. (Item 34) 25. The stem cell of item 24, further comprising modulated expression of one or more MHC-I and MHC-II human leukocyte antigens compared to wild-type stem cells. (Item 35) 35. The stem cell of item 34, wherein the modulated expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II deleted from at least one allele of the cell. (Item 36) 25. The stem cell of item 24, further comprising one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II deleted from at least one allele of the cell. (Item 37) 37. The stem cell according to items 35 to 36, wherein the transcriptional regulator of MHC-I or MHC-II is selected from the group consisting of NLRC5, CIITA and B2M. (Item 38) 37. The stem cell according to items 35 to 36, wherein the transcriptional regulator of MHC-I or MHC-II comprises NLRC5. (Item 39) 37. The stem cell according to items 35 to 36, wherein the transcriptional regulator of MHC-I or MHC-II comprises CIITA. (Item 40) 37. The stem cell according to items 35 to 36, wherein the transcriptional regulator of MHC-I or MHC-II comprises B2M. (Item 41) Human stem cells that do not express NLRC5. (Item 42) NLRC5 - / - 42. The cell of item 41, which is a knockout stem cell. (Item 43) Human stem cells that do not express CIITA. (Item 44) CIITA - / - 44. The cell of item 43, which is a knockout stem cell. (Item 45) Human stem cells that do not express B2M. (Item 46) B2M - / - 46. ​​The cell of item 45, which is a knockout stem cell. (Item 47) A human stem cell that does not express one or more of NLRC5, CIITA and B2M. (Item 48) A human stem cell that does not express one or more of HLA-A, HLA-B and HLA-C. (Item 49) 49. The cell according to items 41 to 48, which expresses one or more tolerogenic factors. (Item 50) 50. The cell of item 49, wherein the tolerogenic factor inhibits immune rejection. (Item 51) 50. The cell of item 49, wherein the tolerogenic factor is inserted at a safe harbor locus of at least one allele of the cell. (Item 52) 52. The cell of item 51, wherein the safe harbor locus comprises the AAVS1 locus. (Item 53) 52. The cell of item 51, wherein the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitory factor, and IL-35. (Item 54) 49. The cell according to items 41 to 48, which is an embryonic stem cell. (Item 55) 49. The cell according to items 41 to 48, which is a pluripotent stem cell. (Item 56) 49. The cell according to items 41 to 48, which is low immunogenic. (Item 57) 49. The cell according to items 41 to 48, which has reduced expression of MHC-I compared to a wild-type human stem cell. (Item 58) 49. The cell according to items 41 to 48, which has reduced expression of MHC-II compared to a wild-type human stem cell. (Item 59) 49. The cell according to items 41 to 48, which has reduced expression of one or more of HLA-A, HLA-B and HLA-C compared to a wild-type human stem cell. (Item 60) A method of using the cells according to items 41 to 48 for cell replacement therapy. (Item 61) A human stem cell comprising one or more tolerogenic factors inserted into a safe harbor locus of at least one allele of said cell. (Item 62) 62. The cell of item 61, wherein the tolerogenic factor inhibits immune rejection. (Item 63) 62. The cell of item 61, wherein the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitory factor, and IL-35. (Item 64) 62. The cell of item 61, wherein the safe harbor locus comprises the AAVS1 locus. (Item 65) 62. The cell of item 61, which does not express one or more of NLRC5, CIITA and B2M. (Item 66) 62. The cell of item 61, which does not express one or more of HLA-A, HLA-B and HLA-C. (Item 67) 62. The cell of item 61, which has reduced expression of one or more of HLA-A, HLA-B and HLA-C compared to a wild-type human stem cell. (Item 68) A method for preparing a hypoimmunogenic stem cell, comprising modulating expression of one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens by stem cells, thereby preparing the hypoimmunogenic stem cell. (Item 69) 1. A method for modulating expression of one or more MHC-I and MHC-II human leukocyte antigens by a stem cell, comprising deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell, thereby modulating expression of the one or more MHC-I and MHC-II human leukocyte antigens. (Item 70) 70. The method of claim 68, wherein said modulating expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises reducing expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens. (Item 71) 70. The method of claim 68, wherein modulating expression of the one or more MHC-I and MHC-II human leukocyte antigens comprises inhibiting expression of the one or more MHC-I and MHC-II human leukocyte antigens. (Item 72) 70. The method of claim 68, wherein modulating expression of the one or more MHC-I and MHC-II human leukocyte antigens comprises disrupting expression of the one or more MHC-I and MHC-II human leukocyte antigens. (Item 73) 69. The method of claim 68, wherein modulating expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell. (Item 74) 74. The method of item 73, wherein the transcriptional regulator of MHC-I or MHC-II is selected from the group consisting of NLRC5, CIITA and B2M. (Item 75) 74. The method of item 73, wherein the transcriptional regulator of MHC-I or MHC-II comprises NLRC5. (Item 76) 74. The method of claim 73, wherein the transcriptional regulator of MHC-I or MHC-II comprises CIITA. (Item 77) 74. The method of item 73, wherein the transcriptional regulator of MHC-I or MHC-II comprises B2M. (Item 78) 70. The method of claim 68, further comprising modulating expression of one or more tolerogenic factors by the stem cells. (Item 79) 79. The method of claim 78, wherein modulating expression of the tolerogenic factor comprises increasing expression of the tolerogenic factor. (Item 80) 80. The method of claim 78, wherein the tolerogenic factor is inserted into a safe harbor locus of at least one allele of the cell. (Item 81) 79. The method of claim 78, wherein the safe harbor locus comprises the AAVS1 locus. (Item 82) 79. The method of claim 78, wherein the tolerogenic factor inhibits immune rejection. (Item 83) 79. The method of claim 78, wherein the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitory factor, and IL-35. (Item 84) 1. A method for preparing hypoimmunogenic stem cells, comprising modulating expression of one or more tolerogenic factors by said stem cells, thereby preparing said hypoimmunogenic stem cells. (Item 85) 85. The method of claim 84, wherein modulating expression of the tolerogenic factor comprises increasing expression of the tolerogenic factor. (Item 86) 85. The method of claim 84, wherein the tolerogenic factor is inserted into a safe harbor locus of at least one allele of the cell. (Item 87) 87. The method of claim 86, wherein the safe harbor locus comprises the AAVS1 locus. (Item 88) 85. The method of item 84, wherein the tolerogenic factor inhibits immune rejection. (Item 89) 85. The method of claim 84, wherein the tolerogenic factor is selected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1 inhibitory factor, and IL-35. (Item 90) 85. The method of claim 84, further comprising modulating expression of one or more MHC-I and MHC-II human leukocyte antigens by the cells. (Item 91) 91. The method of claim 90, wherein modulating expression of the one or more MHC-I human leukocyte antigens and MHC-II human leukocyte antigens comprises deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell. (Item 92) 85. The method of claim 84, further comprising deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell. (Item 93) 93. The method according to items 91 to 92, wherein the transcriptional regulator of MHC-I or MHC-II is selected from the group consisting of NLRC5, CIITA and B2M. (Item 94) 93. The method according to items 91 to 92, wherein the transcriptional regulator of MHC-I or MHC-II comprises NLRC5. (Item 95) 93. The method according to items 91 to 92, wherein the transcriptional regulator of MHC-I or MHC-II comprises CIITA. (Item 96) 93. The method according to items 91 to 92, wherein the transcriptional regulator of MHC-I or MHC-II comprises B2M. (Item 97) A hypoimmunogenic stem cell comprising a modified genome, the hypoimmunogenic stem cell comprising a first genomic modification in which the NLRC5 gene has been edited to reduce or eliminate NLRC5 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239. (Item 98) A hypoimmunogenic stem cell comprising a modified genome, the hypoimmunogenic stem cell comprising a first genome modification in which the CIITA gene has been edited to reduce or eliminate CIITA surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352. (Item 99) A hypoimmunogenic stem cell comprising a modified genome, the hypoimmunogenic stem cell comprising a first genomic modification in which a B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240 to 85644. (Item 100) A hypoimmunogenic stem cell comprising a modified genome, (a) a first genomic modification in which the NLRC5 gene is edited to reduce or eliminate NLRC5 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353 to 81239; (b) a second genome modification in which the CIITA gene is edited to reduce or eliminate CIITA surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352; and / or (c) A third genome modification in which the B2M gene is edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240 to 85644. Hypoimmunogenic stem cells comprising: (Item 101) A hypoimmunogenic stem cell comprising a modified genome, wherein the NLRC5 gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating NLRC5 surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239. (Item 102) A hypoimmunogenic stem cell comprising a modified genome, wherein the CIITA gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CIITA surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184-36352. (Item 103) A hypoimmunogenic stem cell comprising a modified genome, wherein a B2M gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating B2M surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644. (Item 104) A hypoimmunogenic stem cell comprising a modified genome, (a) a first genomic modification in which the NLRC5 gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating NLRC5 surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239; (b) a second genomic modification in which the CIITA gene is edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CIITA surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA is deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352; and / or (c) a third genomic modification in which the B2M gene has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating B2M surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a first pair of a Cas protein or a nucleic acid encoding a Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644. Low immunogenic stem cells. (Item 105) 1. A method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in the cell. (Item 106) A method for producing hypoimmunogenic stem cells, the method comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell. (Item 107) 1. A method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in the cell. (Item 108) 1. A method for producing hypoimmunogenic stem cells, comprising: (a) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding said Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing said NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in said cell; (b) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a second pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell; and / or (c) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a third pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 81240 to 85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in the cell. A method comprising: (Item 109) 1. A method for producing hypoimmunogenic stem cells, the method comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding said Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in said cells. (Item 110) A method for producing hypoimmunogenic stem cells, the method comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in the cell. (Item 111) 1. A method for producing hypoimmunogenic stem cells, comprising contacting a stem cell with a Cas protein or a nucleic acid sequence encoding said Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in said cells. (Item 112) 1. A method for producing hypoimmunogenic stem cells, comprising: (a) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding said Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 36353-81239, thereby editing said NLRC5 gene to reduce or eliminate NLRC5 surface expression and / or activity in said cell; (b) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding said Cas protein, and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 5184 to 36352, thereby editing the CIITA gene to reduce or eliminate CIITA surface expression and / or activity in said cell; and / or (c) contacting a stem cell with a Cas protein or a nucleic acid sequence encoding said Cas protein, and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 81240 to 85644, thereby editing the B2M gene to reduce or eliminate B2M surface expression and / or activity in said cell. A method comprising:

Claims

[Claim 1] The invention as depicted in the drawings.

Images