Targeted non-viral DNA insertions

Abstract

To provide methods and compositions for editing the genome of a cell.SOLUTION: Provided is a method of editing the genome of a cell, the method comprising: (a) providing a Cas9 ribonucleoprotein complex (RNP)-DNA template complex including: (i) the RNP, wherein the RNP comprises a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and wherein the Cas9 nuclease domain cleaves the target region to create an insertion site in the genome of the cell; and (ii) a double-stranded or single-stranded DNA template, wherein the size of the DNA template is greater than about 200 nucleotides, wherein the 5' and 3 ' ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking the insertion site, and wherein the molar ratio of RNP to DNA template in the complex is from about 3 : 1 to about 100: 1; and (b) introducing the RNP-DNA template complex into the cell.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Related prior applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 520,117, filed June 15, 2017, and U.S. Provisional Patent Application No. 62 / 522,180, filed August 30, 2017, both of which are incorporated herein by reference in their entireties.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. P50 GM082250 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention The ability to introduce small mutations (indels) into targeted sites in a cell's genome by electroporating a Cas9-gRNA complex (RNP) into the cell has been developed. However, because these mutations are random and introduced by non-homologous end joining, these modifications can knock proteins out of frame (Schumann et al. PNAS 112(33): 10437-10442 (2015)). Other methods have been developed to introduce predetermined DNA sequences into designated target sites in the genome by electroporating small, chemically synthesized, single-stranded DNA oligonucleotides (ssODNs). This is less efficient than NHEJ, but allows for the integration of very small amounts of exogenous DNA (usually about 1 to 30 base pairs) by homology-directed repair (HDR), which allows the final sequence to be determined. However, the size of these oligonucleotides is limited to the length of DNA that can be chemically synthesized (<approximately 200 bp), a large portion of which is occupied by homologous arms, making this method unusable for many applications due to the limited integration size. In addition to size limitations, it is well documented that electroporation of naked DNA into cells, especially naked DNA larger than approximately 200 bp, often leads to massive cell death due to activation of innate cellular defense mechanisms (Cornu et al. Nat. Med. 23: 415-423 (2017) (Non-Patent Document 2); Hornung and Latz, Nature Reviews Immunology 10: 123-130 (2010) (Non-Patent Document 3); Zhao et al., Mol. Ther. 13(1): 151-159 (2006) (Non-Patent Document 4)). Non-integrating viral vectors, such as integrase-deficient lentiviral vectors or adeno-associated viral (AAV) vectors, have been used to deliver large donor nucleic acid sequences into cells, but these vectors require viral infection and can result in off-target effects.Therefore, there is a need for compositions and methods for the targeted insertion of large nucleotide sequences into the genome of a cell. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Schumann et al. PNAS 112(33): 10437-10442 (2015) [Non-patent document 2] Cornu et al. Nat. Med. 23: 415-423 (2017) [Non-patent document 3] Hornung and Latz, Nature Reviews Immunology 10: 123-130 (2010) [Non-patent document 4] Zhao et al., Mol. Ther. 13(1): 151-159 (2006) Summary of the Invention

[0005] The present invention relates to compositions and methods for editing the genome of a cell. The present inventors have found that large nucleotide sequences, for example, sequences with a length of more than about 200 nucleotides, can be inserted into a targeted region in the genome of a cell. In some methods, the integration of sequences with a length of more than about 200 nucleotides occurs while reducing off-target effects and / or reducing the loss of cell viability.

[0006]

[0006] In some embodiments, the present invention provides methods for editing the genome of a cell, comprising: (a) providing a Cas9 ribonucleoprotein complex (RNP)-DNA template complex, the RNP-DNA template complex comprising: (i) an RNP comprising a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and the Cas9 nuclease domain cleaves the target region to generate an insertion site in the genome of the cell; and (ii) a double-stranded or single-stranded DNA template, wherein the DNA template is greater than about 200 nucleotides in size, and wherein the 5' and 3' ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences adjacent to the insertion site, wherein the molar ratio of RNP:DNA template in the complex is about 3:1 to about 100:1; and (b) introducing the RNP-DNA template complex into the cell.

[0007] In some embodiments, the DNA template is a linear DNA template. In some instances, the DNA template is a single-stranded DNA template. In certain embodiments, the single-stranded DNA template is a pure single-stranded DNA template.

[0008] In some embodiments, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for about 1 minute to about 30 minutes at a temperature of about 20-25° C. In some embodiments, the RNP-DNA template complex and cells are mixed prior to introducing the RNP-DNA template complex into the cells.

[0009] In some embodiments, the RNP comprises a Cas9 nuclease. In some embodiments, the RNP comprises a Cas9 nickase. In some embodiments, the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. In some embodiments, the at least two structurally distinct RNP complexes comprise structurally distinct Cas9 nuclease domains. In some embodiments, the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs. In some embodiments, when the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs, each structurally distinct RNP complex comprises a Cas9 nickase, and the structurally distinct guide RNAs hybridize to opposite strands of the target region.

[0010] In some embodiments, the step of introducing the RNP-DNA template complex into the cell comprises electroporation. In some embodiments, the molar ratio of RNP:DNA template is about 5:1 to about 15:1. In some embodiments, the molar ratio of RNP:DNA template is about 5:1 to about 10:1. In some embodiments, the molar ratio of RNP:DNA template is about 8:1 to about 12:1. In some embodiments, the DNA template is at a concentration of about 2.5 pM to about 25 pM. In some embodiments, the size of the DNA template is greater than about 1 kb. In some embodiments, the amount of DNA template is about 1 μg to about 10 μg.

[0011] In some embodiments, the RNP-DNA template complex is about 1 x 10 5 pieces ~ approx. 2×10 6 In some embodiments, the cells are primary hematopoietic cells or primary hematopoietic stem cells. In some embodiments, the primary hematopoietic cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + CD8 + T cells. [The present invention 1001] (a) providing a Cas9 ribonucleoprotein complex (RNP)-DNA template complex, the RNP-DNA template complex comprising: (i) an RNP comprising a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes to a target region of the genome of a cell, and wherein the Cas9 nuclease domain cleaves the target region to generate an insertion site in the genome of the cell; (ii) a double-stranded or single-stranded DNA template, the DNA template being greater than about 200 nucleotides in size, and the 5' and 3' ends of the DNA template comprising nucleotide sequences that are homologous to genomic sequences adjacent to the insertion site; wherein the molar ratio of RNP:DNA template in the complex is from about 3:1 to about 100:1; and (b) introducing the RNP-DNA template complex into a cell A method for editing the genome of a cell, comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the RNP-DNA template complex is formed by incubating the RNP with the DNA template at a temperature of about 20-25° C. for a period of about less than 1 minute to about 30 minutes. [The present invention 1003] 1003. The method of any one of claims 1001 to 1002, wherein the DNA template is a linear DNA template. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the DNA template is a single-stranded DNA template. [The present invention 1005] 1004. The method of any one of claims 1001 to 1003, wherein the DNA template is a pure single-stranded DNA template. [The present invention 1006] The method of any of claims 1001 to 1005, wherein the RNP-DNA template complex and the cell are mixed prior to introducing the RNP-DNA template complex into the cell. [The present invention 1007] 1007. The method of any one of claims 1001 to 1006, wherein the RNP comprises a Cas9 nuclease. [The present invention 1008] 1007. The method of any one of claims 1001 to 1006, wherein the RNP comprises a Cas9 nickase. [The present invention 1009] 1009. The method of any of claims 1001 to 1008, wherein the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. [The present invention 1010] 1009. The method of claim 10, wherein the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs. [The present invention 1011] 1010. The method of claim 10, wherein the structurally distinct RNP complexes each comprise a Cas9 nickase and the structurally distinct guide RNAs hybridize to opposite strands of the target region. [The present invention 1012] 1009. The method of claim 10, wherein the at least two structurally distinct RNP complexes comprise structurally distinct Cas9 nuclease domains. [The present invention 1013] The method of any one of claims 1001 to 1012, wherein the introducing step comprises electroporation. [The present invention 1014] 1014. The method of any of claims 1001 to 1013, wherein the molar ratio of RNP:DNA template is from about 5:1 to about 15:1. [The present invention 1015] 15. The method of any one of claims 1001 to 1014, wherein the molar ratio of RNP:DNA template is from about 5:1 to about 10:1. [The present invention 1016] 16. The method of any of claims 1001 to 1015, wherein the molar ratio of RNP:DNA template is from about 8:1 to about 12:1. [The present invention 1017] 17. The method of any one of claims 1001 to 1016, wherein the DNA template is greater than about 1 kb in size. [The present invention 1018] 8. The method of any one of claims 1001 to 1017, wherein the DNA template is at a concentration of about 2.5 pM to about 25 pM. [The present invention 1019] The method of any one of claims 1001 to 1018, wherein the amount of the DNA template is about 1 µg to about 10 µg. [The present invention 1020] The method of any of claims 1001 to 1019, wherein the cells are primary hematopoietic cells or primary hematopoietic stem cells. [The present invention 1021] RNP-DNA template complexes were approximately 1 × 10 5 pieces ~ approx. 2×10 6 The method according to any one of claims 1001 to 1020, wherein the vector is introduced into a cell. [The present invention 1022] The method of any one of claims 1001 to 1021, wherein the cells are primary hematopoietic cells. [The present invention 1023] The method of claim 1022, wherein the primary hematopoietic cells are immune cells. [The present invention 1024] The method of claim 1023, wherein the immune cell is a T cell. [The present invention 1025] The method of claim 1024, wherein the T cell is a regulatory T cell, an effector T cell, or a naive T cell. [The present invention 1026] Regulatory T cells, effector T cells, or naive T cells are CD4 + The method of claim 1025, wherein the T cell is a T cell. [The present invention 1027] T cells are CD8 + The method of claim 1024, wherein the T cell is a T cell. [The present invention 1028] T cells are CD4 + CD8 + The method of claim 1024, wherein the T cell is a T cell. [Brief explanation of the drawings]

[0012] This application includes the following drawings. The drawings are intended to illustrate certain aspects and / or features of the compositions and methods and to supplement any description of the compositions and methods. The drawings do not limit the scope of the compositions and methods unless the written description expressly dictates otherwise.

[0013] (Figure 1) Shows low cell viability after electroporation of high concentrations of naked DNA required to achieve workable editing efficiency in cells. (Figure 2) We demonstrate that complexing of the DNA template (plasmid) with RNPs during electroporation by a brief incubation at room temperature before adding cells reduces the loss of viability typically seen when a fixed amount of long plasmid dsDNA is electroporated. (Figure 3) We demonstrate that complexing of DNA templates (linear double-stranded DNA (dsDNA) templates) with RNPs during electroporation by a brief incubation at room temperature before adding cells reduces the loss of viability typically seen when long linear dsDNA templates are electroporated. (Figure 4) An exemplary molar ratio of RNP:DNA template of approximately 10:1 maintains both integration efficiency and viability after electroporation. (Figure 5) An exemplary molar ratio of RNP:DNA template of approximately 10:1 balances the effects of viability loss and efficiency, maximizing the number of positive cell integrations. (Figure 6) An exemplary molar ratio of RNP:DNA template of approximately 10:1 allows for highly efficient insertion of large templates, greater than approximately 750 base pairs in size. (Figure 7) Shows that insertion of long DNA templates can still result in a certain amount of off-target integration. (Figure 8) We demonstrate that off-target integration can be reduced by using a long single-stranded DNA (ssDNA) template as the donor. (Figure 9) Shows that two gRNAs and Cas9 nickase (D10A) can be used to insert non-viral integration as disclosed herein, which prevents off-target dsDNA cleavage. (Figure 10) Figures 10A-F show that co-electroporation of CRISPR / Cas9 RNP reduces dsDNA-induced viability loss. (A) Linear dsDNA templates (homologous recombination repair templates, approximately 1350 bp in length, targeting GFP fusion to RAB11A, Figure 11A) electroporated into primary human T cells result in significant viability loss with increasing template amounts. Electroporation of the same amount of dsDNA template with 100 pmol of RNP surprisingly increased viability. (B) For both plasmid and linear dsDNA templates, the addition of RNP increased viability after electroporation. Notably, no loss of viability was observed with short ssDNA oligodonor nucleotides (ssODNs). (C) To observe increased viability, RNP must be delivered simultaneously with DNA. T cells from two donors were electroporated twice, with an 8-hour break between each. Although closely spaced electroporations resulted in a high degree of cell death, RNP and linear dsDNA templates could be delivered separately. However, initial RNP electroporation did not enhance survival when the DNA template was electroporated later compared with cells receiving DNA first and then RNP. (D–F) Assuming that RNP and DNA must be delivered simultaneously, we assayed whether additional simultaneous preincubation before electroporation would further enhance survival. Increasing preincubation times (0 to 15 min) did not result in any difference in survival, but surprisingly, viability was enhanced when RNP and cells were mixed first and the DNA template was added immediately before electroporation (RNP+ cells; +HDRT) (E). However, in wells where RNP and DNA HDR template were mixed before adding cells (RNP+HDRT; + cells), a sharp increase in the proportion of HDR (GFP+ cells) was observed, regardless of how long the RNP and DNA template were preincubated. Viability was measured 2 days after electroporation, and GFP expression was measured on day 4. Graphs (B, D, F) show data from two healthy donors. (Figure 11) Figures 11A-F demonstrate the development of efficient large non-viral gene targeting. (A) Systematic analysis of the effects of cell culture and stimulation conditions, RNP and DNA template formulations, and electroporation conditions using a 96-well high-throughput electroporator allowed for the rapid optimization of both cell viability (total number of live cells in the culture) and HDR efficiency (% of GFP-positive cells). (B) Schematic diagram (not to scale) of a long (1350 bp) linear dsDNA template encoding a GFP sequence flanked by regions homologous to the N-terminus of the housekeeping gene RAB11A. Upon induction of a dsDNA break at the N-terminus of RAB11A, the GFP sequence can be seamlessly introduced by homology-directed repair (HDR) to generate an endogenously labeled RAB11A-GFP fusion protein. (C) Primary human T cells were cultured for 2 days using various combinations of T cell receptor (TCR) stimulation and cytokines, followed by electroporation of RAB11A-targeted RNP and HDR template, followed by culture under various culture conditions for 5 days post-electroporation. (D) Of the RNP and HDR template concentrations tested here, optimal GFP insertion into RAB11A was achieved at intermediate reagent concentrations. Further testing (Figure 16) narrowed the optimal concentrations to 50 pmol of RNP and 4 μg of dsDNA HDRT. (E) Array testing of electroporation pulse conditions showed that conditions that generally yielded higher HDR efficiency reduced viability. EH115 was selected to optimize HDR while maintaining sufficient viability. (F) Using the parameters optimized in C–D, primary human CD4 + and CD8 + Highly efficient insertion of GFP into the endogenous RAB11A gene was achieved by non-viral targeting in T cells. Viability and efficiency were assayed 3 days (E) or 5 days (C, D, and F) after electroporation. Individual points represent the mean + standard deviation (E) for individual blood donors (C and D) or two individual donors. Green highlights indicate the conditions ultimately selected for the non-viral gene targeting protocol. (Figure 12) Figures 12A-B show that non-viral gene targeting enables rapid and efficient genetic manipulation in primary human T cells. (A) Timeline of non-viral gene targeting. Approximately one week is required to design, order, and assemble any novel combination of genome editing reagents (gRNA and homology-directed repair template). Two days before electroporation, primary human T cells (Figure 15) isolated from blood or various other sources are stimulated. dsDNA HDR templates can be easily generated by PCR, followed by SPRI purification to achieve a highly enriched product suitable for electroporation. On the day of electroporation, the RNP-complexed gRNA, HDR template, and harvested, stimulated T cells are mixed and electroporated (a process that takes approximately one and a half hours). After electroporation, engineered T cells can be easily expanded for an additional two weeks. (B) Viability refers to the percentage of viable cells relative to an equivalent population (no-electroporation control) that underwent all protocol steps except for the actual electroporation. Unless otherwise noted, the decrease in viable cells after electroporation was empirically determined to occur after 2 days, and all viability measurements were recorded at that time point. The term "efficiency" refers to the percentage of viable cells in the culture that express the "knocked-in" exogenous sequence (e.g., GFP). Finally, the total number of cells positive for the desired integration was calculated by multiplying the efficiency by the absolute cell number. Methodological changes that maximized efficiency were often suboptimal for the total number of positive cells, and methodological changes that maximized the total number of positive cells were often suboptimal for efficiency. (Figure 13) Figures 13A-D show optimization of primary human T cell stimulation for non-viral gene targeting. (A) Alternative pre-electroporation stimulation conditions were applied for two days prior to electroporation. CD3 / CD28 bead-bound stimulation, along with a cytokine stimulation cocktail of IL-2, IL-7, and IL-15, achieved higher viability, editing rates, and total positive cell counts than plate-bound antibody stimulation. (B) Alternative bead:cell ratios, along with removal of beads prior to electroporation, demonstrated an optimal 1:1 ratio. (C) Non-bead-based CD3 / CD28 / CD2 stimulation resulted in lower editing efficiency than CD3 / CD28 beads at the optimal ratio. (D) Commercially available XVivo15 medium achieved similar viability but higher editing efficiency compared to RPMI. Interestingly, serum-free Immunocult medium also enabled highly efficient editing of human primary CD3+ T cells. Four days after electroporation, the efficiency of GFP insertion (dsDNA RAB11A-GFP HDRT) and absolute counts of total GFP+ cells were performed. Two dots per condition represent values ​​obtained from two healthy blood donors. (Figure 14) Figures 14A-D show optimization of primary human T cell treatment after electroporation. (A) Electroporation of CD3+ T cells from a healthy donor on days 2 or 3 post-stimulation achieved efficient targeted GFP integration. When DNA template was included in both electroporations, double electroporation on both days sharply reduced viability while slightly increasing efficiency (Figure 10). (B) Additional CD3 / CD28 stimulation after electroporation reduced proliferation capacity. (C) High doses of IL-2 after electroporation improved both efficiency and viability. The addition of additional IL-7 and IL-15 did not improve editing, unlike during pre-electroporation stimulation (Figure 13). (D) Culture density after electroporation had only a small effect on insertion efficiency. Four days after electroporation, the efficiency of GFP insertion (dsDNA RAB11A-GFP HDRT) and absolute counts of all GFP+ cells were performed. Two dots per condition represent values ​​obtained from two healthy blood donors. (Figure 15) Figures 15A-B demonstrate efficient non-viral gene targeting in fresh and frozen T cells isolated from multiple sources. (A) A dsDNA RAB11A-GFP HDR template was inserted into both fresh and frozen T cells from two healthy donors. High rates of GFP insertion were observed in both conditions, demonstrating the adaptability of non-viral gene targeting to research or clinical protocols requiring cell freezing. (B) Similarly, highly efficient GFP-targeted integration was observed in primary human CD3+ T cells isolated from whole blood, plasma apheresis residue, and leukapheresis. (Figure 16) Figures 16A-B show the optimization of RNP and HDR template formulations for non-viral gene targeting. (A) Across three donors, a consistent trend emerged: electroporation of increasing amounts of dsDNA HDR template (RAB11A-GFP) increased efficiency while gradually decreasing cell viability, while intermediate concentrations of both HDR template and RNP tested yielded the greatest total number of GFP+ cells. (B) A further targeting optimization series in three additional donors yielded the optimal formulation, in which 4 μg of HDR template was electroporated in parallel with 50 pmol of RNP. Four days after electroporation, the efficiency of GFP insertion and absolute counting of total GFP+ cells were performed. Multiple dots in graph (B) represent technical replicates. (Figure 17) Figures 17A-C show the optimization of electroporation parameters for delivery of large non-viral HDR templates. (A) The raw data shown here is summarized in Figure 11E. Systematic variation of electroporation conditions in a Lonza 4D Nucleofector. The final selected pulse code, EH115, was consistently the most efficient code when using Lonza P3 electroporation buffer. Other alternative codes, such as EO-148, were optimized for total positive cell counts. (B) Validation testing of a subset of electroporation conditions also identified pulse code EO-155 in OMEM buffer as a combination with moderate efficiency but high total positive cell counts. (C) Electroporation of a total volume of 24 μL (RNP + HDRT + cells) made a significant contribution to cell viability and maintained high efficiency. Electroporation volumes greater than 24 μL generally resulted in electroporation failure. Four days after electroporation, the efficiency of dsDNA RAB11A-GFP insertion (A, C) or dsDNA BATF-GFP insertion (B) and absolute counts of total GFP+ cells were performed. (Figure 18) Figures 18A-D demonstrate the diverse applications of nonviral gene targeting in primary human T cells. (A) Highly efficient genomic targeting of multiple endogenous genes in primary human T cells was achieved with GFP-fusion constructs using nonviral HDR templates and corresponding RNPs. (B) Confocal microscopy of live primary human T cells 7 days after electroporation of the indicated HDR templates confirmed the specificity of fusion protein targeting. Scale bars in each image are 5 μm. (C) Nonviral targeting of GFP-fusion constructs to the RAB11A and CD4 genes in bulk human primary T cells. The RAB11A fusion was GFP-positive in both CD4+ and CD8+ cells, whereas the CD4+ fusion was only positive in CD4+ T cells (representative flow cytometry images above, quantification below). (D) Primary human T cells were engineered to express GFP fused to the endogenous transcription factor BATF. Eleven days after electroporation, nuclei were isolated and subjected to the CUT&RUN method. Anti-GFP or anti-BATF antibodies were used to identify GFP-BATF and total BATF chromatin interaction sites. Four days after electroporation, flow cytometry was performed to assay viability and efficiency (A, C, D). Data shown are representative of at least two different donors. (Figure 19) Figures 19A-B show reproducible non-viral gene targeting across targeted loci. (A) Four days after electroporation of one of five different GFP templates and the corresponding RNP into primary CD3+CD8+ T cells from six healthy donors, GFP expression was observed across both templates and donors. Note the consistency of GFP expression levels within GFP-positive cells across donors for each of the five loci (relatively high for TUBA1B and ACTB, relatively low for RAB11A and FBL tags). (B) Graphical summary of the percentage of GFP insertion in (A). (Figure 20) Figures 20A-B show reproducible non-viral gene targeting in a cohort of healthy donors. (A) Optimized conditions developed for non-viral gene targeting in cells from a cohort of 12 healthy donors were used to electroporate a consistent dsDNA RAB11A-GFP HDR template and RNP. Significant variability in GFP insertion rates was observed between individual donors, but all achieved robust GFP incorporation (range 22%-57% in CD8+ T cells). Compared to the no-electroporation control, some GFP expression was observed in cells electroporated with the dsDNA RAB11A-GFP HDR template along with off-target RNP targeting CXCR4. (B) Graphical summary of the GFP insertion rates in (A). Among a cohort of 12 healthy donors, a slightly higher proportion of CD3+CD8+ T cells (mean 42.0%) expressed GFP compared with CD3+CD4+ T cells (mean 35.2%). (Figure 21) Endogenous tagging of the transcription factor BATF for analysis of chromatin occupancy. Heatmap of anti-BATF, anti-GFP, and no antibody for CUT&RUN data obtained from a primary human T cell population electroporated with a GFP-BATF fusion HDR template (untagged cells were not electroporated). Aligned CUT&RUN binding profiles for each sample were centered on the BATF CUT&RUN peak in untagged cells and ordered by BATF peak intensity in untagged cells. (Figure 22) Figures 22A-E show combinatorial nonviral gene targeting. (A) Co-electroporation of HDR templates to form RAB11A-GFP and / or RAB11A-mCherry fusions in primary human T cells. When both templates were introduced simultaneously, a distinct population of double GFP+mCherry+ cells was found, consistent with biallelic targeting. (B) The potential genotype for individual cells in a quadrant was determined by expression of the two fluorophores. The observed level of biallelic integration was higher than that expected by chance (Figure 23) in cells that obtained at least one integration. Individual dots represent replicates of varying combinations of genes encoding fluorescent proteins (GFP+mCherry, GFP+BFP, mCherry+BFP) as well as HDR template amounts (3-6 μg). (C-D) Multiplexed integration of HDR templates at two separate genomic loci in the same primary human T cells. Two micrograms of each template (4 micrograms total per electroporation) were electroporated with 25 pmol of each RNP (50 pmol total). Cells positive for integration at one site (e.g., GFP+) were much more likely to have integration at a second site (e.g., also mCherry+) than cells lacking the first integration. (E) Simultaneous nonviral gene targeting of a large insertion into three different genomic loci. 1.5 μg of each template (4.5 μg total) was electroporated with 20 pmol of each corresponding RNP (60 pmol total). Similar to the two-site multiplexing, cells positive for one integration (mCherry+ in Q-II, GFP+ in Q-III) were more likely to have a second integration (BFP+) compared with none (QI). Cells positive for two integrations (GFP+ and mCherry+, Q-IV) were more likely to have integration of a third gene (BFP+). Below is a bar graph quantification of cells that were single-, double-, and triple-positive for the fluorophores. All fluorescent readouts were performed 4 days after electroporation. Data shown represent at least two different donors, except for panel E (one donor). (Figure 23) Figures 23A-G show modeling and analysis of biallelic HDR integration by insertion of multiple fluorescent proteins into the same locus. (A) Possible cellular phenotypes when two fluorescent proteins are inserted into the same locus. (B) Two of these phenotype populations are readily apparent. Cells without any functional insertion (lower left quadrant, genotype A) should have an NA / NA genotype (in this case, NA indicates the non-HDR allele, including the WT allele and the NHEJ-edited allele). Dual-fluorescent cells (upper right quadrant, genotype E) should have acquired one copy of each template (assuming an autosomal target locus and no off-target integration) and will have a GFP / RFP genotype. The two single-positive populations are mixed between cells heterozygous for the HDR insertion (genotypes B and C) or homozygous for two copies of the same fluorescent template (genotypes D and F). (C) The total proportion of cells with biallelic HDR integration should be the sum of genotypes D, E, and F. While the proportion of cells with genotype E (double fluorophore-positive) is readily apparent from the phenotype, the proportion of cells with genotypes D and F is not. Application of a simple probability model allows deconvolution of multiple genotypes within a single fluorophore-positive phenotype and thus estimation of the true proportion of cells homozygous for HDR. (D) Biallelic HDR analysis applied among various fluorophore substitutions inserted into the RAB11A locus. (E-F) Dual-fluorescent biallelic integrations were observed among the targeted loci. While the total proportion of cells with insertions varied with the efficiency of each target site, the fold enrichment in the observed proportion of homozygous cells over the expected proportion by chance was consistent among loci. (G) Attempted integration of three different fluorophores by HDR into the same locus. Since a maximum of two targeted insertions are possible (in two alleles at that locus; assuming a diploid genome), cells positive for all three loci (triple positive) should not be observed.In fact, there are numerous single-fluorophore incorporations (single-positives) as well as cells positive for various substitutions of two fluorophores (double-positives), but there is a 30-fold decrease in the number of triple-positive cells compared to double-positives. Four days after electroporation, all flow cytometry analyses of fluorescent protein expression were performed. Shown are multiple technical replicates from one (E, F) or two (D, G) healthy donors. Bar graphs show the mean + standard deviation. (Figure 24) Figures 24A-B show multiplexed integration, demonstrating that gaining HDR integration at one locus increases the likelihood of HDR at additional loci. (A) Two HDR template replacements from a set of six dsDNA HDR templates (targeting RAB11A, CD4, and CLTA; each site carries GFP or RFP) were electroporated into CD3+ T cells isolated from a healthy donor. Four days after electroporation with the two indicated HDR templates and their respective on-target RNPs, the percentage of cells positive for each template was analyzed when gating on cells positive or negative for the other template. Not only was dual-template multiplexing possible across diverse template combinations, but gating on cells positive for one template (Template 1+ cells) yielded an enriched population of cells more likely to be positive for the second template compared with cells negative for the first template (Template 1- cells, black). For double-multiplexing experiments, 2 μg of each template was electroporated with 30 pmol of each relevant RNP. (B) Electroporation of an additional template allows for three-site multiplexing using various HDR template combinations. Cells positive for the third template can be further enriched by gating on cells positive for both other templates compared to single-positive cells. Data shown are the mean + standard deviation from multiple technical replicates from two healthy donors. (Figure 25) Figures 25A-F show that D10A nickase and ssDNA HDR templates reduce off-target integration. (A) A combination of Cas9 RNP and a RAB11A-GFP dsDNA HDR template was electroporated into primary human T cells. The dsDNA template alone or with an RNP containing a scrambled gRNA that did not match any sequence in the human genome produced a small but detectable amount of GFP expression, which was significantly increased when the dsDNA template was electroporated with a gRNA targeting a site different from the targeted RAB11A-GFP integration site (the "off-target RNP" targets CXCR4 exon 1). (B) When the RAB11A-GFP dsDNA HDR template was electroporated with the off-target RNP, off-target integration was consistently present in cells from different donors, and less off-target integration occurred when the dsDNA HDR template was electroporated alone. (C) When a single off-target CXCR4 gRNA was used, the Cas9 nuclease mutant D10A (nickase) and inactive dCas9 significantly reduced off-target integration, whereas D10A nickase (together with an "on-target" pair of gRNAs in the PAM-Out orientation) led to efficient on-target integration of the RAB11A dsDNA HDR template. (D) Electroporation of the ssDNA HDR template reduced off-target integration to the limit of detection (comparable to levels without electroporation of the template) both in the absence of added nuclease and in the case of induced off-target dsDNA cleavage (off-target gRNA + Cas9). (E-F) In the case of integration of a GFP fusion at the RAB11A site, the use of D10A nickase and the ssDNA HDR template reduced on-target HDR compared to Cas9 and the dsDNA template, but strongly reduced off-target integration to undetectable levels (GFP integration with the on-target gRNA). All fluorescent readouts were performed 4 days after electroporation.Data shown represent the mean + standard deviation of at least two different donors (A and E) or two different donors (C, D and F). (Figure 26) Figures 26A-D show fluorescence estimation and quantification of off-target integration events among multiple HDR templates. (A) Diagram of HDR-mediated insertion at the N-terminus of the target locus (not to scale). Homology arms specify the exact sequence where the insert (in this case, a GFP tag) will be inserted, allowing for intact integration of the exogenous sequence. When a GFP fusion protein is created, this on-target integration results in GFP fluorescence, dependent on RNP cleavage adjacent to the integration site. (B) Double-stranded DNA can be integrated via homology-independent repair mechanisms at off-target sites, either at spontaneous dsDNA breaks or, possibly, through random integration at induced double-strand breaks, e.g., at off-target RNP cleavage sites. This effect can be exploited to enable targeted integration of dsDNA sequences at desired induced dsDNA breaks (HITIs) in senescent cell types lacking the ability to perform HDR. Importantly, the entire dsDNA template is integrated, including any potential homologous arms. If the homologous arms contain promoter sequences (e.g., for N-terminal fusion tags), these off-target integrations can drive observable expression of the inserted sequence even without the desired correct HDR insertion. (C) Bars represent the observed GFP+ percentage from human CD3+ T cells electroporated with the indicated constructs. Flow cytometry for fluorescent protein expression can be used to rapidly assess functional off-target integration. An increase in the percentage of fluorescent cells above the detection limit when the template alone is electroporated likely represents random integration at spontaneous dsDNA breaks. While not all off-target integrations result in fluorescent protein expression, the relative differences in functional off-target expression between different templates can be assayed. Inclusion of a CXCR4-targeting RNP dramatically increases the observed off-target homology-independent integration, presumably via HITI-type insertion events, with the greatest increase (from 1% to >30% in this donor) occurring through electroporation and HDR-mediated insertion of the correct RNP.(D) Comparison of on-target GFP expression (right column) and functional off-target integration (middle column) among five templates. Average expression (bars) of the two biological donors (dots) is graphed. (Figure 27) Figures 27A-B show GFP expression between HDR templates and gRNA matrices. (A) GFP expression was analyzed in CD3+CD4+ primary human T cells from healthy donors 7 days after electroporation of dsDNA HDR templates and their corresponding gRNA matrices with CXCR4 gRNA and a no-RNP control. As expected with dsDNA templates, off-target integration was observed for all combinations, but the highest GFP expression was observed in on-target conditions for all gRNAs and HDR templates. (B) Heatmap summary of flow cytometry data from (A). One HDR template, a C-terminal GFP fusion tag to nuclear factor FBL, consistently showed high off-target expression for all gRNAs. (Figure 28) Figures 28A-D show efficient HDR in primary human T cells using Cas9 nickase. (A) Diagram of the genomic locus containing the first exon of RAB11A. Using spCas9 with one guide RNA (gRNA 1) with a dsDNA HDR template to integrate GFP in frame with RAB11A immediately after the start codon resulted in efficient GFP expression (Figure 11F). Using Cas9 nickase with two gRNAs (D10A mutant) reduced the possibility of off-target cleavage. (B-C) A series of single gRNAs and dual gRNA combinations were tested for GFP insertion efficiency at the RAB11A N-terminal locus. As expected, when nuclease-inactive Cas9 (dCas9) was used, no gRNAs showed appreciable levels of GFP insertion. When Cas9 was used, multiple single gRNA cleavages flanking the insertion site showed GFP integration, but none were as efficient as gRNA 1. D10A nickase showed little to no GFP integration with single guides, but efficient GFP integration with multiple dual-guide combinations. GFP integration was observed only with gRNA combinations in which the two PAM sequences were oriented away from each other (PAM-Out). (D) Raw data presented in Figure 25C demonstrates low levels of functional off-target integration upon electroporation of an off-target gRNA (targeting CXCR4), likely due to the requirement that D10A nickase have two gRNAs in close association to induce dsDNA cleavage. Dots in all displays (B–D) represent technical replicates in two labeled healthy donors. (Figure 29) Figures 29A-H show reduced Treg frequencies and impaired Treg suppressive capacity in subjects with two loss-of-function IL2RA mutations. (A) CD3+CD4+ T cells from a healthy donor and all family members, including IL2RA heterozygotes with loss-of-function IL2RA mutations (c.530 Het 1, c.800 Het 1-3) and compound heterozygote children (Comp. Het 1-3), were analyzed by flow cytometry to assess the presence of CD25hiCD127lo Tregs. (B) In the healthy donor and single hets, CD4+FoxP3+ T cells are predominantly CD25hiCD127lo. In compound heterozygotes, a CD127loCD4+FoxP3+ population is present but does not express IL2RA. (C) Clinical phenotyping performed at two separate sites confirms that compound heterozygotes have normal frequencies of CD127loFoxP3+ cells. (D) Lack of IL2RA surface expression in compound heterozygote 3 led to aberrant downstream signaling after stimulation with IL-2, but not IL-7 or IL-15, as measured by pStat5 expression. (E) Due to the inability to select CD25hi Tregs from CD25-deficient compound heterozygotes, an alternative gating strategy was established to enrich FoxP3+ cells from CD3+CD4+ T cells using the surface marker CD127loCD45RO+TIGIT+. Intracellular FoxP3 staining from the indicated gated populations is shown. (F) These CD3+CD4+CD127loCD45RO+TIGIT+ potential "Tregs" were highly enriched for FoxP3 and showed some suppressive capacity when cultured with CFSE-labeled stimulated responder T cells (Tresp) from healthy donors, whereas CD3+CD4+CD127loCD45RO+TIGIT+ from compound heterozygotes did not. Stimulated Tresp population (solid curve), unstimulated Tresp (dashed curve). (G) Correction of the CD25 mutation in compound heterozygotes still leaves the other mutations individually, leaving the cells as single heterozygotes.To confirm that such potential corrections result in some level of functional suppression, CD4+CD25hiCD127lo Tregs were isolated from c.530 and c.800 single heterozygote family members and their suppressive capacity was assayed as in (F). (H) Dot plot summary of Treg suppressive capacity in cells from healthy donors, CD25-deficient compound heterozygotes (F), and CD25+ / - c.530 or c.800 heterozygotes (G). CD3+CD4+CD127loCD45RO+TIGIT+ Tregs from compound heterozygotes did not exhibit suppressive capacity, whereas normal CD4+CD25hiCD127lo Tregs from single heterozygote family members exhibited some suppressive capacity, consistent with the lack of a pronounced clinical phenotype compared to compound heterozygotes. (Figure 30) Figures 30A-E show monogenic autoimmune mutations corrected by nonviral gene targeting in primary human T cells. (A) Three siblings in a family carry two different IL2RA (encoding the high-affinity IL-2 receptor CD25) mutations (c.530A>G creates a stop codon in IL2RA exon 4; c.800delA creates a frameshift mutation in IL2RA exon 8 resulting in a run-on of approximately 100 amino acids). (B) These three compound heterozygous littermates show greatly reduced (but not completely absent) cell surface expression of IL2RA on primary T cells. Nonviral gene targeting of the c.530 mutation by electroporation of a Cas9 RNP and dsDNA HDR template containing the correct IL2RA sequence (with a silent mutation in the targeting PAM sequence) successfully rescued IL2RA cell surface expression in a subset of T cells from each compound heterozygous littermate 2 days after electroporation. (C) Seven days after nonviral gene targeting, targeted T cells exhibited increased Stat5 phosphorylation levels upon IL-2 stimulation compared with untargeted controls. (D) Nine days after nonviral gene targeting to correct the c.530 mutation, IL2RA+ T cells from three compound heterozygous donors contained increased levels of FoxP3+ cells compared with untargeted cells or healthy donor cells. (E) Using an optimized therapeutic reagent set (D10A nickase and ssDNA HDR template), nonviral gene targeting and correction of the c.530 mutation is feasible and efficient. After 9 days of ex vivo expansion following electroporation (2 days after restimulation), T cells from one compound heterozygous donor were stained for IL2RA surface expression. (Figure 31) Figures 31A-D show the identification of compound heterozygous mutations in IL2RA and the design of a corrective CRISPR-Cas9 genome-targeting reagent. (A) Initial genetic testing in the proband using an in-house targeted next-generation sequencing multigene panel of over 40 genes known to be involved in monogenic forms of diabetes was negative. Subsequent exome sequencing in the proband and three of his parents revealed two causative mutations in the IL2RA gene. The mother had a mutation in exon 4 of IL2RA. (B) The father had a heterozygous mutation (c.530G>A) in TIFF2025078701000002.tif4128, resulting in a premature stop codon. The proband had one heterozygous mutation (c.800delA) in TIFF2025078701000003.tif4128, resulting in a frameshift mutation that created a 95-amino acid long run-on. Sanger sequencing confirmed that the proband was compound heterozygous for both mutations. (C) Diagram of the IL2RA protein annotated with the approximate locations of the two identified IL2RA mutations. SD1, sushi domain 1; SD2, sushi domain 2; TM, transmembrane; C, cytoplasmic. (D) Genomic sequence containing the indicated mutations (in the case of the c.530G>A allele, TIFF2025078701000004.tif4128 and in the case of the c.800delA allele We designed CRISPR-Cas9 genome-targeting reagents to correct two IL2RA mutations using the IL2RA gene encoding IL2RA 1.25 (TIFF2025078701000005.tif4128). The gRNAs were designed to cut adjacent to the site of each mutation, 8 bp away for the c.530 mutation and 7 bp away for the c.800 mutation. For each mutation, the HDR template ( TIFF2025078701000006.tif4128 and In the case of c.800 A set of 530 RNAs (TIFF2025078701000007.tif4128) was designed, including the modified sequences and silent mutations in the degenerate bases to disrupt the PAM sequence ("NGG") for each guide RNA. TIFF2025078701000008.tif4128 and c.800 The corrected allele + silent PAM disruption sequence for TIFF2025078701000009.tif4128 is shown. Genomic regions (not to scale) for the c.530 mutation site (hg38 ch10:6021526-6021557) and the c.800 mutation site (hg38 ch10:6012886-6012917) are shown. Both ssODN HDR templates (ssDNA with 60-bp homology arms) and large dsDNA or ssDNA HDR templates (with approximately 300-bp homology arms, as shown) were used. (Figure 32) Figures 32A-C show HDR-mediated correction of the IL2RA c.530A>G loss-of-function mutation. (A) Unlike gRNAs targeting the c.800delA mutation in the C-terminus of IL2RA, gRNAs targeting the c.530A>G mutation (which generates a stop codon in an internal exon) resulted in substantial (approximately 90%) knockdown of IL2RA in healthy donors and single heterozygotes (c.800 Het 2 and 3) 2 days after electroporation of RNP alone (blue) into CD3+ T cells. Knockdown was also observed in all three compound heterozygotes, starting with a very small proportion of IL2RA+ cells, presumably because some small amount of protein can be surface expressed from the c.800delA allele. This reduced CD25 expression could be partially rescued by the inclusion of an ssODN HDR template and more substantially rescued using a large dsDNA HDR template. Both template types contained the corrected sequence, a silent mutation to remove the gRNA PAM sequence, and 60-bp (ssODN) or approximately 300-bp (large dsDNA) homology arms (Figure 32). Unlike targeting the c.800delA mutation for correction, CD25 surface expression in T cells from compound heterozygotes was only observed when the HDR template was included. In all three compound heterozygotes, the dsDNA HDR template elicited a greater percentage of CD25+ cells. (B) Increased pStat5 signaling in response to IL-2 stimulation (200 U / mL) 7 days after electroporation in CD3+ T cells from a compound heterozygote patient undergoing HDR-mediated mutation correction compared to no-electroporation or RNP-only controls. (C) Similarly, in the compound heterozygote patient, an increased percentage of CD25+FoxP3+ cells was observed 9 days after electroporation under HDR-correction conditions. When the c.530 mutation was targeted for HDR-correction in compound heterozygote 3, a lower percentage of correction was observed, likely due to altered cellular conditions related to the patient's disease or their immunosuppressant drug regimen. Electroporation was performed according to the optimized non-viral genome-targeting protocol described in the Examples.For ssODN electroporation, 100 pmol in 1 μL of H2O was electroporated. (Figure 33) Figures 33A-C show non-HDR-mediated correction of the IL2RA c.800delA frameshift loss-of-function mutation. (A) Histograms of CD25 surface expression in CD3+ T cells in all children of a family with two loss-of-function IL2RA mutations, including three compound heterozygotes expressing minimal amounts of IL2RA on the surface (no electroporation, gray). Two days after electroporation of RNPs containing gRNAs directed against one of the two mutations, a one-base-pair deletion in the final exon of IL2RA (c.800delA) that results in a run-on through the normal stop codon, CD3+ T cells from a healthy donor and single hets (c.800 Het 2 and 3) show a slight increase in CD25- cells (RNP only, blue). The low knockout is likely due to gRNAs targeting the C-terminus of the protein, where small indels can cause a relatively insignificant loss of surface protein expression. Strikingly, RNP alone resulted in CD25 surface expression in nearly 50% of edited T cells in all three compound heterozygotes. An increase in the percentage of cells with CD25 correction compared to RNP alone could be achieved by including the ssODN HDR template sequence carrying the mutation correction (RNP + ssODN, purple) and could be further increased when a longer dsDNA HDR template was used to correct the mutation (RNP + dsDNA HDRT, green) (Figure 32). (B) Phospho pStat5 signaling in edited CD3+ T cells in response to high-dose IL-2 stimulation (200 U / mL) after 7 days of expansion after electroporation. The increased number of pStat5+ cells correlated with increased CD25 surface expression (A). (C) After 9 days of growth after electroporation, intracellular FoxP3 staining reveals a dramatic increase in the percentage of CD25+FoxP3+ cells among CD3+ T cells compared to controls without electroporation (approximating the percentage of CD25+FoxP3+ cells found in similarly cultured healthy donors). Electroporation was performed according to the optimized non-viral genome targeting protocol (Examples). For ssODN electroporation, 100 pmol were electroporated in 1 μL of HO. (Figure 34) Figures 34A-B show reduced HDR capacity and altered clinical phenotype in compound heterozygous IL2RA loss-of-function patients receiving immunosuppressive drugs. (A) Flow cytometry analysis of GFP expression 6 days after electroporation of the positive HDR control RAB11A-GFP dsDNA HDR template into CD3+ T cells from the indicated patients revealed lower GFP expression in three compound heterozygotes compared with their two c.800 heterozygote littermates. Compared to a cohort of 12 similarly edited healthy donors (Figure 20), the c.800 heterozygotes and compounds het 1 and 2 were within the typical range observed among healthy donors, while compound het 3 showed lower GFP expression than any of the healthy donors analyzed. Notably, in compound het 3, HDR-mediated correction of the c.530 mutation was substantially lower than in the other two compound hets (Figure 31A), yet CD25 surface expression was similar after electroporation of c.800delA-targeting RNP alone. Unlike HDR-mediated repair, NHEJ-mediated frameshift correction at c.800delA may not require cell proliferation, consistent with compound het 3 being the only compound heterozygous patient on effective immunosuppressive medication at the time of blood collection and T cell isolation. (B) Altered cellular status in relation to the patient's disease could also contribute to the reduced HDR rate. TIGIT and CTLA4 expression levels were measured by flow cytometry in unedited CD4+ T cells isolated from each patient. Consistent with an altered activation state, cells from compound het 3 displayed a distinct phenotype, with increased TIGIT and CTLA4 expression compared with the healthy donor, heterozygous family members, and two other compound heterozygous littermates. (Figure 35) Figures 35A-B show several methods for generating long ssDNA HDR templates. (A) If we can generate large enough quantities of long single-stranded DNA sequences for electroporation, we can reduce off-target integration without excessively compromising on-target efficiency. One method involves a two-step selective exonuclease digestion that specifically degrades one strand of the PCR product, which is labeled by a 5' phosphorylation that is easily added to the PCR primer before amplification. (B) We also applied a second ssDNA generation method based on sequential in vitro transcription (IVT) and reverse transcription (RT) reactions. The PCR product with an added short T7 promoter serves as the IVT template for generating ssRNA products. After annealing of the RT primer and reverse transcription, an RNA / DNA hybrid is formed, which can be easily converted into a long ssDNA template by incubation in sodium hydroxide (which selectively degrades the RNA strand). (C) Two days after electroporation, viability in CD3+ T cells electroporated with ssDNA template alone was higher than that in CD3+ T cells electroporated with dsDNA template alone (Figure 11). (D) The ssDNA RAB11A-GFP HDR template exhibited high efficiency of GFP integration similar to the dsDNA template and maintained high integration efficiency at higher molar template amounts, likely due to increased viability (C) and a lower DNA template mass per molar. Each dot represents at least two healthy donors (C, D). DETAILED DESCRIPTION OF THE INVENTION

[0014] definition As used in this specification and the appended claims, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0015] The terms "nucleic acid" and "nucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term "nucleic acid" is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0016] The term "gene" can refer to a segment of DNA involved in making or encoding a polypeptide chain. A gene can include regions before and after the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" can refer to a segment of DNA involved in making or encoding a non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., small guide RNA), or microRNA.

[0017] "Treating" refers to any indication of successful treatment or amelioration or prevention of a disease, condition, or disorder, including any objective or subjective parameter, such as relief; remission; a decrease in symptoms or making the disease state more tolerable to the patient; a slowing of the rate of degeneration or decline; or a less debilitating end point of degeneration. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a medical examination. Thus, the term "treating" includes the administration of a compound or agent of the present invention to prevent, delay, alleviate, arrest, or inhibit the onset of symptoms or conditions associated with the diseases, conditions, or disorders described herein. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject. "Treating" or "treatment" using the methods of the invention includes preventing the onset of symptoms in a subject who may be at risk for a disease or disorder associated with a disease, condition, or disorder described herein but who has not yet experienced or displayed symptoms, suppressing (delaying or arresting) the symptoms of a disease or disorder, providing relief from symptoms or side effects of a disease (including palliative treatment), and alleviating (causing regression) symptoms of a disease. Treatment can be prophylactic (to prevent or delay the onset of a disease or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of a disease or condition. As used herein, the term "treatment" includes preventative (e.g., prophylactic), curative, or palliative treatment.

[0018] A "promoter" is defined as one or more nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter also optionally includes distal enhancer or repressor elements, which can be located several thousand base pairs from the transcription start site.

[0019] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0020] As used herein, the terms "complementary" or "complementarity" refer to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U) and G and C.

[0021] As used throughout this specification, "subject" refers to an individual. For example, a subject may be a mammal, such as a primate, more specifically a human. Non-human primates are also subjects. The term "subject" includes domesticated animals, such as cats and dogs, livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary and medical uses and formulations are contemplated herein. The term does not denote a particular age or sex. Thus, adult or newborn subjects, regardless of gender, are considered to be encompassed. As used herein, "patient" or "subject" may be used interchangeably and may refer to a subject suffering from a disease or disorder.

[0022] The term "CRISPR / Cas" refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of Eubacteria and Archaea organisms. CRISPR / Cas systems include type I, II, and III subtypes. Wild-type type II CRISPR / Cas systems utilize the RNA-mediated nuclease Cas9 complexed with guide and activator RNAs to recognize and cleave foreign nucleic acids. Guide RNAs with both guide RNA and activator RNA activity are also known in the art. In some cases, such dual-activity guide RNAs are referred to as small guide RNAs (sgRNAs).

[0023] Cas9 homologs are found in a wide variety of eubacteria, including bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologs are described, for example, in Chylinksi, et al., RNA Biol. 2013 May 1; 10(5):726-737; Nat. Rev. Microbiol. 2011 June; 9(6):467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep 24; 110(39):15644-9; Sampson et al., Nature. 2013 May 9; 497(7448):254-7; and Jinek, et al., Science. 2012 Aug 17; 337(6096):816-21. The Cas9 nuclease domain can be optimized for efficient activity or enhanced stability in host cells.

[0024] As used herein, the term "Cas9" refers to an RNA-mediated nuclease (e.g., of bacterial or archaeal origin or derived therefrom). Exemplary RNA-mediated nucleases include the Cas9 protein and their homologs, including, but not limited to, CPF1 (see, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015). Similarly, as used herein, the term "Cas9 ribonucleoprotein" complex, etc., refers to a complex of a Cas9 protein and a crRNA (e.g., a guide RNA or a small guide RNA), a complex of a Cas9 protein and a trans-activating crRNA (tracrRNA), a complex of a Cas9 protein and a small guide RNA, or a combination thereof (e.g., a complex comprising a Cas9 protein, a tracrRNA, and a crRNA guide RNA).

[0025] The term "editing" as used herein in relation to editing the genome of a cell refers to inducing structural changes in the sequence of a genome in a target genome region.For example, editing can take the form of inserting a nucleotide sequence into the genome of a cell.The nucleotide sequence can encode a polypeptide or a fragment thereof.Such editing can be carried out by inducing a double-strand break in the target genome region, or by inducing a pair of single-strand nicks on opposite strands on either side of the target genome region.Methods for inducing single-strand or double-strand breaks in or within a target genome region include using a Cas9 nuclease domain or its derivative and a guide RNA or a pair of guide RNAs directed to the target genome region.

[0026] The term "introduce" as used herein in relation to the introduction of an RNP-DNA template complex refers to the transfer of the RNP-DNA template complex from the outside of the cell to the inside of the cell. In some cases, introduction refers to the transfer of the RNP-DNA template complex from the outside of the cell to the nucleus of the cell. Various methods of such transfer are possible, including electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, transfer via cell-penetrating peptides, liposome-mediated transfer, etc.

[0027] As used herein, the term "heterologous" refers to something that is not normally found in nature. The term "heterologous sequence" refers to a sequence that is not normally found in a given cell in nature. As such, a heterologous nucleotide or protein sequence can be (a) foreign to its host cell (i.e., exogenous to the cell); (b) one that is naturally found in the host cell (i.e., endogenous) but is present in the cell in an unnatural amount (i.e., greater or less than naturally found in the host cell); or (c) one that is naturally found in the host cell but located outside its native locus.

[0028] The term "primary" as used herein with respect to primary cells or primary stem cells refers to cells that are neither transformed nor immortalized. Such primary cells can be cultured, subcultured, or passaged a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from organisms, systems, organs, or tissues, optionally sorted, and used directly without culture or subculture. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contact with (e.g., culture in the presence of) CD3, CD28 agonist, IL-2, IFN-γ, or a combination thereof.

[0029] As used herein, the phrase "hematopoietic stem cells" refers to a type of stem cell that can give rise to blood cells. Hematopoietic stem cells can give rise to cells of the myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are primarily found in the bone marrow, but can also be isolated from peripheral blood or a portion thereof. Various cell surface markers can be used to identify, select, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are identified by the c-kit + and lin - In some cases, human hematopoietic stem cells are identified as CD34 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, human hematopoietic stem cells are identified as CD34 - , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, human hematopoietic stem cells express CD133 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + , lin - In some cases, mouse hematopoietic stem cells are identified as CD34 lo / - , SCA-1 + , Thy1 + / lo , CD38 + , C-kit + , lin - In some cases, hematopoietic stem cells express CD150 + CD48 - CD244 - is.

[0030] As used herein, the phrase "hematopoietic cells" refers to cells derived from hematopoietic stem cells. Hematopoietic cells can be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood or a portion thereof). Alternatively, hematopoietic cells can be obtained or provided by isolating hematopoietic stem cells and differentiating the stem cells. Hematopoietic cells include cells with limited capacity to differentiate into additional cell types. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-committed progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocytic-erythroid progenitor cells. Hematopoietic cells include lymphoid and myeloid cells, such as lymphocytes, erythrocytes, granulocytes, monocytes, and platelets. In some embodiments, the hematopoietic cells are immune cells, such as T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells. In some embodiments, the cells are innate immune cells.

[0031] As used herein, the phrase "T cells" refers to lymphoid cells that express T cell receptor molecules. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells are CD4 + , CD8 + or CD4 + and CD8 + T cells can be helper cells, such as type T h 1. T h 2. T h 3. T h 9. T h 17 or T FH The T cells can be cytotoxic T cells. The regulatory T cells can be FOXP3 + or FOXP3 - The T cells can be alpha / beta T cells or gamma / delta T cells. In some cases, the T cells are CD4 + CD25hi CD127 lo In some cases, the T cells are regulatory T cells selected from the group consisting of Tr1, Th3, CD8+CD28-, Treg17, and Qa-1-restricted T cells, or a combination or subpopulation thereof. In some cases, the T cells are FOXP3 + T cells. In some cases, T cells are CD4 + CD25 lo CD127 hi effector T cells. In some cases, T cells are CD4 + CD25 lo CD127 hi CD45RA hi CD45RO - These are naive T cells.

[0032] The T cell can be a genetically engineered recombinant T cell. In some cases, the recombinant T cell has a recombinant (e.g., mutant or heterologous) T cell receptor or chimeric antigen receptor (CAR). For example, the T cell receptor can have one or more mutations in the complementarity-determining regions of the T cell receptor to alter antigen specificity. As another example, the T cell receptor can be mutated (e.g., within the endodomain) to increase or decrease signaling. As yet another example, the T cell receptor can be replaced with a heterologous T cell receptor. As yet another example, the T cell receptor can be replaced with a polypeptide, such as an antibody or antibody fragment, having a different receptor domain. In some cases, the T cell receptor is a chimeric receptor comprising a targeting domain (e.g., an antibody fragment), a transmembrane domain, and an intracellular or endodomain domain. The endodomain can comprise one or more signaling domains and / or adapter domains to provide robust T cell activation and anti-antigen activity.

[0033] As used herein, the term "non-homologous end joining" or NHEJ refers to a cellular process in which the broken or nicked ends of a DNA strand are directly joined without the need for a homologous template nucleic acid.NHEJ can result in the addition, deletion, substitution, or combination thereof of one or more nucleotides at the repair site.

[0034] As used herein, the term "homologous recombination repair (HDR)" refers to the cellular process in which the broken or nick end of DNA strand is repaired by polymerization from homologous template nucleic acid.Therefore, the original sequence is replaced with the sequence of the template.The homologous template nucleic acid can be provided by the homologous sequence (sister chromatid, homologous chromosome, or repeat region on the same or different chromosome) elsewhere in the genome.Alternatively, exogenous template nucleic acid can be introduced to obtain specific HDR-induced changes in the sequence at target site.In this way, specific mutations can be introduced at the break site.

[0035] As used herein, single-stranded DNA template or double-stranded DNA template refers to the DNA oligonucleotide that cell can use as template for HDR.Generally, single-stranded DNA template or double-stranded DNA template has at least one homologous region with target site.In some cases, single-stranded DNA template or double-stranded DNA template has two homologous regions on both sides of the region that contains the heterologous sequence that is inserted into target cleavage site.

[0036] Detailed Description of the Invention The following description describes various aspects and embodiments of the present compositions and methods. The specific embodiments are not intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least within the scope of the disclosed compositions and methods. This description should be read from the perspective of a person skilled in the art. Therefore, it does not necessarily include information that is well known to a person skilled in the art.

[0037] Provided herein are compositions and methods for editing the genome of a cell.The present inventors have surprisingly found that in the absence of a viral vector, large nucleotide sequences, for example, nucleotide sequences with a length of more than about 200 nucleotides or base pairs, can be inserted into the genome of a cell.In some embodiments, in the absence of a viral vector, nucleotide sequences with a length of more than about 200 nucleotides or base pairs can be inserted into the genome of primary immune cells.

[0038] The integration of large nucleic acid, for example, nucleic acid of size greater than 200 nucleotides, into cells can be limited by low integration efficiency, off-target effect and / or loss of cell viability.Described herein are methods and compositions for achieving the integration of nucleotide sequence, for example, nucleotide sequence of size greater than about 200 nucleotides, into the genome of cells.In some methods, the efficiency of integration is increased, off-target effect is reduced, and / or loss of cell viability is reduced.

[0039] method A method for editing the genome of a cell can include: (a) providing a Cas9 ribonucleoprotein complex (RNP)-DNA template complex, the RNP-DNA template complex comprising: (i) an RNP comprising a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and the Cas9 nuclease domain cleaves the target region to generate an insertion site in the genome of the cell; and (ii) a double-stranded or single-stranded DNA template, wherein the DNA template is greater than about 200 nucleotides in size, and wherein the 5' and 3' ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences adjacent to the insertion site, wherein the molar ratio of RNP:DNA template in the complex is about 3:1 to about 100:1; and (b) introducing the RNP-DNA template complex into the cell.

[0040] In some embodiments, the methods described herein provide a delivery efficiency of RNP-DNA template complexes of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99%, or higher. In some cases, the efficiency is determined in terms of cells that remain viable after introduction of the RNP-DNA template into the cells. In some cases, the efficiency is determined in terms of the total number of cells (viable or nonviable) into which the RNP-DNA template is introduced.

[0041] As another example, delivery efficiency can be determined by quantifying the number of genome-edited cells in a cell population (compared to the total cells or total viable cells obtained after the introduction step).Various methods can be used to quantify genome editing.These methods include, but are not limited to, using mismatch-specific nucleases, such as T7 endonuclease I; sequencing one or more target loci (for example, by Sanger sequencing of cloned target locus amplified fragments); and high-throughput deep sequencing.

[0042] In some embodiments, the loss of cell viability is reduced compared to the loss of cell viability after naked DNA is introduced into cells or after DNA is introduced into cells using a viral vector.The reduction can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage therebetween.In some embodiments, the off-target effects of integration are reduced compared to the off-target integration after naked DNA is introduced into cells or after DNA is introduced into cells using a viral vector.The reduction can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage therebetween.

[0043] In some cases, the methods described herein provide high viability of cells into which the RNP-DNA template has been introduced. In some cases, the viability of cells into which the RNP-DNA template has been introduced is at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99% or higher. In some cases, the viability of cells into which the RNP-DNA template has been introduced is about 20% to about 99%, about 30% to about 90%, about 35% to about 85% or 90% or more, about 40% to about 85% or 90% or more, about 50% to about 85% or 90% or more, about 50% to about 85% or 90% or more, about 60% to about 85% or 90% or more, or about 70% to about 85% or 90% or more.

[0044] In the methods provided herein, the molar ratio of RNP:DNA template can be from about 3:1 to about 100:1. For example, the molar ratio can be from about 5:1 to 10:1, from about 5:1 to about 15:1, from 5:1 to about 20:1, from 5:1 to about 25:1, from about 8:1 to about 12:1, from about 8:1 to about 15:1, from about 8:1 to about 20:1, or from about 8:1 to about 25:1.

[0045] In some embodiments, the DNA template is at a concentration of about 2.5 pM to about 25 pM. For example, the DNA template concentration can be about 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 pM, or any concentration therebetween. In some embodiments, the size or length of the DNA template is about 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2.0 kb, 2.1 kb, 2.2 kb, Larger than 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4.0kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8kb, 4.9kb, 5.0kb or any DNA template size in between these sizes. For example, the size of the DNA template can be about 200 bp to about 500 bp, about 200 bp to about 750 bp, about 200 bp to about 1 kb, about 200 bp to about 1.5 kb, about 200 bp to about 2.0 kb, about 200 bp to about 2.5 kb, about 200 bp to about 3.0 kb, about 200 bp to about 3.5 kb, about 200 bp to about 4.0 kb, about 200 bp to about 4.5 kb, or about 200 bp to about 5.0 kb. In some embodiments, the amount of DNA template is about 1 μg to about 10 μg. For example, the amount of DNA template can be about 1 μg to about 2 μg, about 1 μg to about 3 μg, about 1 μg to about 4 μg, about 1 μg to about 5 μg, about 1 μg to about 6 μg, about 1 μg to about 7 μg, about 1 μg to about 8 μg, about 1 μg to about 9 μg, or about 1 μg to about 10 μg.In some embodiments, the amount of DNA template is about 2 μg to about 3 μg, about 2 μg to about 4 μg, about 2 μg to about 5 μg, about 2 μg to about 6 μg, about 2 μg to about 7 μg, about 2 μg to about 8 μg, about 2 μg to about 9 μg, or 2 μg to about 10 μg. In some embodiments, the amount of DNA template is about 3 μg to about 4 μg, about 3 μg to about 5 μg, about 3 μg to about 6 μg, about 3 μg to about 7 μg, about 3 μg to about 8 μg, about 3 μg to about 9 μg, or about 3 μg to about 10 μg. In some embodiments, the amount of DNA template is about 4 μg to about 5 μg, about 4 μg to about 6 μg, about 4 μg to about 7 μg, about 4 μg to about 8 μg, about 4 μg to about 9 μg, or about 4 μg to about 10 μg. In some embodiments, the amount of DNA template is about 5 μg to about 6 μg, about 5 μg to about 7 μg, about 5 μg to about 8 μg, about 5 μg to about 9 μg, or about 5 μg to about 10 μg. In some embodiments, the amount of DNA template is about 6 μg to about 7 μg, about 6 μg to about 8 μg, about 6 μg to about 9 μg, or about 6 μg to about 10 μg. In some embodiments, the amount of DNA template is about 7 μg to about 8 μg, about 7 μg to about 9 μg, or about 7 μg to about 10 μg. In some embodiments, the amount of DNA template is about 8 μg to about 9 μg, or about 8 μg to about 10 μg. In some embodiments, the amount of DNA template is about 9 μg to about 10 μg. In some cases, the size of the DNA template is large enough and in sufficient quantity to be lethal as naked DNA. In some embodiments, the DNA template encodes a heterologous protein or a fragment thereof. In some embodiments, the DNA template comprises regulatory sequences, eg, promoter and / or enhancer sequences, for controlling expression of the heterologous protein or fragment thereof after insertion into the genome of the cell.

[0046] In some cases, DNA template is linear DNA template.In some cases, DNA template is single-stranded DNA template.In some cases, single-stranded DNA template is pure single-stranded DNA template.As used herein, " pure single-stranded DNA " refers to single-stranded DNA that is substantially devoid of the other or opposite strand of DNA." Substantially devoid " refers to pure single-stranded DNA that is devoid of one strand of DNA at least 100 times more than the other strand.

[0047] In some cases, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than about 1 minute to about 30 minutes at a temperature of about 20° C. to about 25° C. For example, the RNP can be incubated with the DNA template at a temperature of about 20° C., 21° C., 22° C., 23° C., 24° C., or 25° C. for about 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, or 30 minutes, or any time in between. In another example, the RNP can be incubated with the DNA template for less than about 1 minute to about 1 minute, less than about 1 minute to about 5 minutes, less than about 1 minute to about 10 minutes, about 5 to 10 minutes, about 5 to 15 minutes, about 10 to about 15 minutes, about 10 to about 20 minutes, or about 10 to about 30 minutes at a temperature of about 20° C. to about 25° C. In some embodiments, the RNP-DNA template complex and cells are mixed before introducing the RNP-DNA template complex into the cells.

[0048] In some embodiments, the step of introducing the RNP-DNA template complex comprises electroporation. Methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in the Examples herein. Further or alternative methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in WO / 2006 / 001614 or Kim, JA et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Further or alternative methods, compositions, and devices for electroporating cells to introduce the RNP-DNA template complex can include those described in U.S. Patent Application Publication Nos. 2006 / 0094095; 2005 / 0064596; or 2006 / 0087522. Further or alternative methods, compositions, and devices for electroporating cells to introduce RNP-DNA template complexes can include those described in Li, LH et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Patent Nos. 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6485961; 7029916; and U.S. Patent Application Publication Nos. 2014 / 0017213; and 2012 / 0088842. Further or alternative methods, compositions, and devices for electroporating cells to introduce RNP-DNA template complexes can include those described in Geng, T. et al., J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010).

[0049] In some embodiments, the Cas9 protein can be in an active endonuclease form, and when bound to a target nucleic acid as part of a complex with a guide RNA or a DNA template, a double-strand break is introduced into the target nucleic acid. The double-strand break can be repaired by NHEJ to introduce random mutations, or by HDR to introduce specific mutations. Various Cas9 nucleases can be used in the methods described herein. For example, a Cas9 nuclease that requires an NGG protospacer adjacent motif (PAM) immediately 3' of the region targeted by the guide RNA can be used. Such a Cas9 nuclease can be targeted to any region of the genome that contains an NGG sequence. As another example, a Cas9 protein that requires an orthogonal PAM motif can be used to target a sequence that does not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, CFP1, those described in Nature Methods 10, 1116-1121 (2013), and those described in Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015.

[0050] In some cases, Cas9 protein is a nickase, and when it binds to a target nucleic acid as part of a complex with a guide RNA, it introduces a single-strand break or nick into the target nucleic acid.A pair of Cas9 nickases, each bound to a structurally different guide RNA, can target two adjacent sites in the target genome region, and thus introduce a pair of adjacent single-strand breaks into the target genome region.Nickase pairs can provide enhanced specificity, because off-target effects are likely to cause a single nick, which is generally repaired without damage by base excision repair mechanisms.Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations.

[0051] In some embodiments, the RNP comprises a Cas9 nuclease. In some embodiments, the RNP comprises a Cas9 nickase. In some embodiments, the RNP-DNA template complex comprises at least two structurally distinct RNP complexes. In some embodiments, the at least two structurally distinct RNP complexes comprise structurally distinct Cas9 nuclease domains. In some embodiments, the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs. In some embodiments, when the at least two structurally distinct RNP complexes comprise structurally distinct guide RNAs, each structurally distinct RNP complex comprises a Cas9 nickase, and the structurally distinct guide RNAs hybridize to opposite strands of the target region.

[0052] In some cases, multiple RNP-DNA templates comprising structurally distinct ribonucleoprotein complexes are introduced into a cell, e.g., a Cas9 protein can be complexed with multiple (e.g., 2, 3, 4, 5, or more, e.g., 2-10, 5-100, 20-100) structurally distinct guide RNAs to target insertion of the DNA template at multiple structurally distinct target genomic regions.

[0053] In the methods and compositions provided herein, the cells include, but are not limited to, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, etc. Optionally, the cells are mammalian cells, e.g., human cells. The cells can be in vitro, ex vivo, or in vivo cells. The cells can also be primary cells, germ cells, stem cells, or progenitor cells. Progenitor cells can be, for example, pluripotent stem cells or hematopoietic stem cells. In some embodiments, the cells are primary hematopoietic cells or primary hematopoietic stem cells. In some embodiments, the primary hematopoietic cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are CD4 + In some embodiments, the T cells are CD8+ In some embodiments, the T cells are CD4 + CD8 + In some embodiments, the T cells are CD4 - CD8 - The modified T cells are also provided. Also provided are any populations of cells modified by any of the methods described herein. In some embodiments, the method further comprises expanding the population of modified cells.

[0054] In some cases, cells are removed from a subject, modified using any of the methods described herein, and administered to the patient. In other cases, any of the constructs described herein are delivered to the patient in vivo. See, for example, U.S. Patent No. 9,737,604 and Zhang et al. "Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy," NPG Asia Materials Volume 9, page e441 (2017).

[0055] In some embodiments, the RNP-DNA template complex is about 1 x 10 5 pieces ~ approx. 2×10 6 For example, the RNP-DNA template complex is introduced into approximately 1 × 10 cells. 5 pieces~approx. 5×10 5 cells, approximately 1 x 10 5 pieces~approx. 1×10 6 pieces, 1×10 5 pieces~approx. 1.5×10 6 pieces, 1×10 5 pieces ~ approx. 2×10 6 pieces, about 1×10 6 pieces~approx. 1.5×10 6 cells or approximately 1 x 10 6 pieces ~ approx. 2×10 6 It can be introduced into individual cells.

[0056] In some cases, the methods and compositions described herein can be used to generate, modify, use, or control recombinant T cells, such as chimeric antigen receptor T cells (CAR T cells). Such CAR T cells can be used to treat or prevent cancer, infectious diseases, or autoimmune diseases in a subject. For example, in some embodiments, one or more gene products are inserted or knocked into T cells to express a heterologous protein (e.g., a chimeric antigen receptor (CAR)).

[0057] composition Also provided herein are a plurality of cells, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of whose genomes comprise targeted insertion of a heterologous DNA template, wherein the DNA template is at least about 200 bp in size. In some embodiments, the plurality of cells comprises primary hematopoietic cells or primary hematopoietic stem cells. In some embodiments, the primary hematopoietic cells are immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are CD8 + In some embodiments, the T cells are CD4 + CD8 + T cells.

[0058] Disclosed are materials, compositions, and components that can be used for, can be used with, can be used to prepare, or are the products of the disclosed methods and compositions. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even if specific reference to each of the various individual and collective combinations and permutations of these compounds is not explicitly disclosed. For example, if a method is disclosed and discussed, and a number of modifications that can be made to one or more molecules, including those in the method, are discussed, unless otherwise indicated, all combinations and permutations of the method and possible modifications are specifically contemplated. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including the steps of methods that use the disclosed compositions. Thus, if there are a variety of additional steps that may be performed, it will be understood that each of those additional steps can be performed with any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0059] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety. [Example]

[0060] The following examples are offered by way of illustration only, not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same or similar results.

[0061] Example I The data provided in Example I was generated as outlined in the following protocol. Established clinical protocol / donor consent • PBMC isolation was performed using SepMate with the manufacturer's protocol. Bulk T cell isolation was performed using EasySep with the manufacturer's protocol. • Freezing was performed using Bambanker medium according to the manufacturer's protocol. ○20 million cells per mL ● Thawing One mL of Roswell Park Memorial Institute Medium (RPMI) was added on top of the thawed cells, which were then combined in the medium and washed. Before stimulation, cells were left in the medium overnight.

[0062] Primary T cell culture Culture medium ○RPMI + 10% FBS ○XVivo15+5% FBS; or ○Immunocult (serum-free) ■ Useful for cell culture in a serum-free environment ●Stimulation 1:1 CD3 / CD28 magnetic Dynabeads ■ Ratios from 0.25:1 to 2:1 can be used. ■ Removal of magnetic beads before electroporation can improve efficiency Cytokines ■Before electroporation ■ 200U / mL IL-2 (essential) ■ 5ng / mL IL-7 (non-essential) ■ 5ng / mL IL-15 (non-essential) After electroporation ■ 500U / mL IL-2 (essential) ●Culture density Before electroporation ■ 1 x 10 cells per 1 mL of culture medium 6pieces Typically, 1 mL goes into a 24-well plate, 30 mL into a T75 flask, or 70 mL into a T175 flask. After electroporation ■ Day 0 - 0.25 10 6 Place electroporated cells in 200 μL of medium into one well of a 96-well round-bottom plate. Day 2 - Add 100 μL of fresh medium to the wells from above (cytokines at 3x concentration after electroporation). Day 4 - The cells were transferred to 500 μL of medium in a 48-well plate with fresh cytokines for further growth, and then split every 2–3 days, adding fresh cytokines each time, until the cells reached approximately 1 × 10 cells per ml of culture. 6 Maintain individuality

[0063] RNP production Mix 160 μM crRNA with 160 μM tracrRNA in a 1:1 ratio. Store the aliquoted stock at -80°C. ■ Resuspend the lyophilized RNA in Tris-HCl (pH 7.4) with 150 mM MgCl Purchase crRNA and tracrRNA from Dharmacon or IDT, and always use tracrRNA from the respective manufacturer with that crRNA. Incubate at 37°C for 30 minutes. ○ Prepare 80 μM gRNA Mix 80µM gRNA with 40µM Cas9 in a 1:1 ratio. Mix the tube by gently tapping the side until the Cas9 precipitate dissolves. Incubate at 37°C for 15 minutes. ○ Make 20 μM RNP RNPs can be used immediately, stored at 4°C for short periods until use, or stored at -80°C for long periods and then thawed before use.

[0064] Homologous recombination repair template (HDRT) generation ● Construction HDRT sequences were constructed using Gibson Assemblies from PCR products and GeneBlocks (IDT), and the final HDRT containing 5' and 3' homology arms and the desired insert was placed into a cloning vector for further propagation. ●Production Linear dsDNA HDRT sequences were generated by high-output PCR amplification (Kapa Hotstart polymerase). PCR amplicons were SPRI purified and concentrated to a final volume of 4 μL of HO per 100 μL of PCR reaction input. HDRT concentrations were analyzed by Nanodrop at a 1:20 dilution. Purity was assayed by gel electrophoresis

[0065] Primary T cell electroporation Electroporation parameters Cell count - 1,000,000 (as few as 200,000 or as many as 2,000,000 work well) Cell volume - 20 μL (this volume can vary between about 10 μL and about 20 μL) ■ Cells are spun at 90G for 10 minutes, aspirated, resuspended in electroporation buffer, and immediately electroporated. Electroporation buffer P3 Alternate buffers, including P2 and OMEM, work well, but the optimal pulse code is different. Buffer P2 produces higher viability and lower efficiency, but the total number of positive cells is similar. ■ OMEM buffer with optimal pulse code (EO155) produces similar viability and efficiency as P3 with optimal pulse code ○RNP amount - Approx. 0.5μL (50pmol) ■ Works well with as little as 1 μL (20 pmol) or as much as 5 μL (100 pmol) ■ The optimal amount of RNP varies with the amount of HDRT, but an exemplary molar ratio of RNP:HDRT of about 10:1 works well. ○HDRT amount - approx. 1μL ■ The volume can vary between about 0.5 μL and about 2 μL. ○HDRT total amount - approx. 5 pmol ■ Smaller and larger amounts are possible, but with different efficiencies Total electroporation volume: approx. 24 μL ○Pulse Code - EH115 ■ Many other pulse codes are possible, but EH115 proved to be the most efficient.

[0066] Electroporation Protocol First, HDRT was dispensed into the wells of a 96-well polypropylene V-bottom plate corresponding to the wells of a 96-well electroporation plate. The indicated RNPs were then added to a 96-well polypropylene V-bottom plate. HDRT and RNP were incubated together at room temperature for 5 minutes. ■Even if it's only 30 seconds, there's no difference in effectiveness. ■ It is important to incubate HDRT and RNP together before adding cells. Finally, the cells were resuspended in electroporation buffer, and 20 μL of cells were added to each well of a 96-well polypropylene V-bottom plate and mixed with the HDRT and RNP already in the well by pipetting up and down three times. For electroporation, 24 μL of the cell + RNP + HDRT mixture was transferred from each well into the corresponding well of a 96-well electroporation plate. Post-electroporation treatment Immediately after electroporation, 80 μL of prewarmed medium was added to each well of the electroporation plate. The plate was incubated in an incubator at 37°C for 15 minutes. ■ When no incubation was performed after electroporation, the efficiency was slightly lower. Incubation for approximately 15 to 60 minutes is possible without loss of efficiency. Cells were transferred from the electroporation plate to the culture plate at the above density. ■ Generally, the electroporation plate was divided into four identical 96-well round-bottom plates pre-filled with medium and cytokines.

[0067] result It would be useful to generate longer DNA constructs, for example by using PCR to generate linear dsDNA constructs that allow for large insert sizes (>1 kb). While this can be done at high throughput, until the present invention, this was not possible because the introduction of DNA is highly toxic, resulting in significant cell death. As shown in Figure 1, the concentration of naked DNA required to achieve viable editing efficiencies results in very low cell viability, making this method impractical.

[0068] Complexing long DNA templates with RNPs rescues cell viability We found that when electroporating long dsDNA (plasmid or linear dsDNA) in amounts sufficient to cause extensive cell death, complexing the DNA with RNP to form an RNP-DNA template complex (by a brief incubation at room temperature prior to addition of cells during electroporation) reduced viability loss. This was true for both plasmid (Figure 2) and linear dsDNA templates (Figure 3). As the amount of electroporated DNA increased, the amount of RNP also increased to maintain viability (Figure 3).

[0069] Cas9 to DNA template ratio for viability and integration efficiency A molar ratio of approximately 10:1 RNP:DNA template maintained both integration efficiency and viability after electroporation (Figure 4). However, ratios ranging from 3:1 to approximately 100:1 also worked well. The use of an RNP:DNA template ratio of approximately 10:1 balanced the effects of loss of viability and efficiency, achieving the greatest number of positive integration cells (Figure 5). This ratio also allowed for highly efficient insertion of large templates (>750 bp) (Figure 6).

[0070] dsDNA templates have some off-target integration that is reduced using ssDNA templates Insertion of long DNA templates can result in small amounts of off-target integration (Figure 7), similar to that seen when using AAV as a donor template. However, some of the methods provided herein use long ssDNA templates as donors, which results in reduced off-target integration (Figure 8).

[0071] Use of Cas9 nickase prevents off-target dsDNA cleavage Another problem besides off-target integration is the off-target dsDNA breaks introduced by Cas9 (which can be repaired as mutations by NHEJ). As shown herein, the highly efficient non-viral integration disclosed herein can be inserted using two gRNAs and Cas9 nickase (D10A) (Figure 9), which prevents off-target dsDNA breaks.

[0072] Example II Isolation of human primary T cells for gene targeting Primary human T cells were isolated from healthy donors using fresh whole blood samples, residue from the leukapheresis chamber after Trima Apheresis (Blood Centers of the Pacific), or leukapheresis products (StemCell). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by Ficoll centrifugation using SepMate tubes (STEMCELL, as per manufacturer's instructions). T cells were isolated from PBMCs from all cell sources by magnetic negative selection using the EasySep Human T Cell Isolation Kit (STEMCELL, as per manufacturer's instructions). Unless otherwise noted, isolated T cells were stimulated and used directly (fresh). When frozen cells were used, previously isolated T cells, frozen in Bambanker freezing medium (Bulldog Bio) as per manufacturer's instructions, were thawed, cultured in unstimulated medium for 1 day, and then stimulated and processed as described for freshly isolated samples. Fresh healthy blood donors provided consent under a protocol approved by the UCSF Committee on Human Research (CHR), and patient samples for gene editing were obtained under a protocol approved by the Yale Internal Review Board (IRB).

[0073] Primary T cell culture Unless otherwise noted, bulk T cells were cultured in XVivo15 medium (STEMCELL) containing 5% fetal bovine serum, 50 mM 2-mercaptoethanol, and 10 mM N-acetyl-L-cystine. Supplement-free serum-free medium (ImmunoCult XF T Cell Expansion Medium, STEMCELL) and RPMI + 10% FBS were used in the indicated experiments (Figure 15). Immediately after isolation, T cells were stimulated for 2 days with anti-human CD3 / CD28 magnetic Dynabeads (ThermoFisher) (bead:cell ratio 1:1) and a cytokine cocktail of 200 U / mL IL-2 (UCSF Pharmacy), 5 ng / mL IL-7 (ThermoFisher), and 5 ng / mL IL-15 (LifeTech). After electroporation, T cells were cultured in medium containing 500 U / mL IL-2. T cells were maintained at a density of approximately 1 million cells per mL of medium throughout culture. Every 2–3 days after electroporation, additional medium was added along with additional fresh IL-2 to a final concentration of 500 U / mL, and cells were transferred to larger culture vessels as needed to maintain a density of 1 million cells / mL.

[0074] RNP production RNPs were generated by annealing binary gRNA to Cas9 as previously described (7, 16). Briefly, crRNA and tracrRNA were chemically synthesized (Dharmacon, IDT), and recombinant Cas9-NLS, D10A-NLS, or dCas9-NLS were recombinantly produced and purified (QB3 Macrolab). Lyophilized RNA was resuspended at a concentration of 160 μM in Tris-HCl (pH 7.4) containing 150 mM KCl and stored in aliquots at -80°C. The crRNA and tracrRNA aliquots were thawed, mixed in a 1:1 volume ratio, and incubated at 37°C for 30 min to form an 80 μM gRNA solution. Recombinant Cas9 and mutants stored at 40 μM in 20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 10% glycerol, and 1 mM DTT were then mixed with 80 μM gRNA at a volume ratio of 1:1 (gRNA:Cas9 molar ratio of 2:1) for 15 minutes at 37° C. to form 20 μM RNPs. RNPs were typically electroporated immediately after complexation.

[0075] dsDNA HDRT production Double-stranded DNA HDRT sequences were generated from PCR products. Novel HDR sequences were constructed by placing HDR template sequences consisting of homologous arms (typically synthesized as gBlocks from IDT) and the desired insert (e.g., GFP) into a cloning vector using Gibson Assemblies for sequence verification and future propagation. These plasmids were used as templates for high-output PCR amplification (Kapa Hotstart polymerase). PCR amplicons (dsDNA HDRT) were SPRI-purified (1.0x) and eluted in a final volume of 3 μL of HO per 100 μL of PCR reaction input. The concentration of HDRT was analyzed by Nanodrop at a 1:20 dilution. The size of the amplified HDRT was confirmed by gel electrophoresis in a 1.0% agarose gel.

[0076] ssDNA HDRT generation by exonuclease digestion To generate long ssDNA as an HDR donor, the DNA of interest was amplified by PCR using one normal, unmodified PCR primer and a second, phosphorylated PCR primer. The DNA strand amplified using the phosphorylated primer becomes the strand degraded using this method. This allows each phosphorylated PCR primer to be used to prepare single-stranded sense or single-stranded antisense DNA. To generate the ssDNA strand of interest, the phosphorylated strand of the PCR product was degraded by subsequent treatment with two enzymes, Strandase Mix A and Strandase Mix B, at 37°C for 5 minutes (per kb). The enzymes were inactivated by incubation at 80°C for 5 minutes. The resulting ssDNA HDR template was SPRI-purified (1.0x) and eluted in HO. A more detailed protocol for the Guide-it™ Long ssDNA Production System (Takara Bio USA, Inc. #632644) can be found on the manufacturer's website.

[0077] ssDNA HDRT synthesis by retrosynthesis As described previously, the ssDNA donor was synthesized by reverse transcription of the RNA intermediate followed by hydrolysis of the RNA strand in the resulting RNA:DNA hybrid product. Briefly, the desired HDR donor was first cloned downstream of a T7 promoter, and the T7-HDR donor sequence was amplified by PCR. RNA was synthesized by in vitro transcription using HiScribe T7 RNA polymerase (New England Biolabs) and reverse transcribed using TGIRT-III (InGex). After reverse transcription, NaOH and EDTA were added to 0.2 M and 0.1 M, respectively, and RNA hydrolysis was carried out at 95 °C for 10 min. The reaction was quenched with HCl, and the final ssDNA product was purified using Ampure XP magnetic beads (Beckman Coulter) and eluted in sterile RNase-free HO. The quality of the ssDNA was analyzed by capillary electrophoresis (Bioanalyzer, Agilent).

[0078] Primary T cell electroporation Two days after initial T cell stimulation, RNP and HDR templates were electroporated. T cells were harvested from the culture vessel and magnetic CD3 / CD28 Dynabeads were removed by placing the cells on a magnet for 2 minutes. Immediately prior to electroporation, the bead-removed cells were centrifuged at 90xg for 10 minutes, aspirated, and resuspended in Lonza electroporation buffer P3 at 20 µL of buffer per million cells. For optimal editing, 1 million T cells per well were electroporated using a Lonza 4D 96-well electroporation system with pulse code EH115. Alternative cell concentrations from 200,000 to 2 million cells per well demonstrated lower efficiency. Alternative electroporation buffers were used as indicated, but the optimal pulse settings were different (EO155 for OMEM buffer). Unless otherwise indicated, 2.5 μL of RNP (50 pmol total) was electroporated with 2 μL of 2 μg / μL HDR template (4 μg total HDR template).

[0079] The order of addition of cells, RNP, and HDRT appeared to be important (Figure 10). For 96-well experiments, HDRT was first dispensed into wells of a 96-well polypropylene V-bottom plate. RNP was then added to HDRT and incubated together at room temperature for at least 30 seconds. Finally, cells resuspended in electroporation buffer were added and quickly mixed with HDRT and RNP by pipetting, and a total volume of 24 μL (cells + RNP + HDRT) was transferred into a 96-well electroporation cuvette plate. Immediately after electroporation, 80 μL of prewarmed medium (without cytokines) was added to each well, and the cells were left in the electroporation cuvette and placed in a cell culture incubator at 37°C for 15 minutes. After 15 minutes, the cells were transferred to the final culture vessel.

[0080] Flow cytometry Flow cytometry analysis was performed on an Attune NxT Accustic Focusing Cytometer (ThermoFisher). Surface staining was performed for CD3-APC-eFluor 780 (SK7, eBiosciences), CD4-PerCP (SK3, Tonbo), CD8-PE-Cy7 (SK1, BD), and IL2RA / CD25-APC (BC96, Tonbo). Intracellular phosphorylation staining was performed using pStat5(Y694)-PacBlue (clone 47, BD). Intracellular cytokine staining for FoxP3 was performed using FoxP3-AF488 (206D, Biolegend).

[0081] Confocal microscopy Drop-cast 10 μl of the live T cell suspension onto a 3 x 1 inch microscope slide and place a 25 mm 2 Samples were prepared by placing a coverslip. Imaging was performed on an upright Nikon A1r laser scanning confocal microscope. Excitation was achieved with a 488 nm OBIS laser (Coherent). A long-working-distance (LWD) 60× Plan Apo 1.20 NA water immersion objective was used, with additional digital zoom achieved by NIS-Elements software. Images were acquired under the "Galvano" mirror setting with 2× line averaging enabled, exported as TIFF, and analyzed with FIJI (ImageJ, NIH).

[0082] CUT&RUN Eleven days after electroporation and four days after restimulation with anti-CD3 / anti-CD28 beads (untagged cells were not electroporated), CUT&RUN was performed on epitope-tagged primary human T cells. Approximately 20% and 10% of electroporated cells showed GFP-BATF expression as determined by flow cytometry in donor 1 and donor 2 samples, respectively. CUT&RUN was performed using anti-GFP (ab290), anti-BATF (sc-100974), and rabbit anti-mouse (ab46540) antibodies as described (18). Briefly, 6 million cells (30 million cells for anti-GFP CUT&RUN on GFP-BATF-containing cells) were collected and washed. Nuclei were isolated and incubated with primary antibody (GFP or BATF) for 2 hours at 4°C with rotation. BATF CUT&RUN samples were further incubated with rabbit anti-mouse antibody for 1 hour. Nuclei were then incubated with Protein A-micrococcal nuclease (kindly provided by the Henikoff lab) for 1 hour at 4°C. Nuclei were equilibrated to 0°C, and MNase digestion proceeded for 30 minutes. Solubilized chromatin CUT&RUN fragments were isolated and purified. Paired-end sequencing libraries were prepared and run on an Illumina Nextseq machine. Sequencing data were processed as described in Skene and Henikoff, "An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites," Elife 6 (2017) doi: 10.7554 / eLife.21856. Reads mapping to centromeres were excluded for peak calling and heatmap generation.

[0083] TLA sequencing and analysis The aforementioned 16 TLA sequencing was performed by Cergentis, as described above. 16Data analysis of integration sites and transgene fusions was performed by Cergentis as described in. TLA sequencing was performed on two healthy donors whose RAB11A locus was edited with dsDNA or ssDNA HDR templates to incorporate GFP fusions. Sequencing reads showing evidence of primer dimers or primer bias (i.e., >99% of observed reads came from a single primer set) were excluded.

[0084] In vitro Treg suppression assay CD4 T cells were isolated using the EasySep Human CD4+ T Cell Enrichment Kit (STEMCELL Technologies). + T cells were enriched. CD3+CD4+CD127loCD45RO+TIGIT+ Treg-enriched cells from IL2RA-deficient and HD subjects, as well as CD3+CD4+CD25hiCD127lo Tregs from CD25+ / - individuals, were sorted by flow cytometry. CD3+CD4+CD25-CD127+ responder T cells (Tresp) were labeled with 5 μM CellTrace CFSE (Invitrogen). Tregs and HD Tresp were co-cultured at a 1:1 ratio in the presence of beads loaded with anti-CD2, anti-CD3, and anti-CD28 (Treg Suppression Inspector; Miltenyi Biotec) at a ratio of 1 bead:1 cell. On days 3.5–4.5, co-cultures were analyzed by FACS for CFSE dilution. % suppression was calculated using the following formula: 1 – (% proliferation with Tregs / % proliferation of Tresp stimulated without Tregs).

[0085] Sorting of corrected Tregs and TSDR analysis Ex vivo expanded Treg and T effector cells from healthy controls and patients with IL2RA compound heterozygous mutations (D6) were thawed and stained. Live cells were sorted based on expression of CD25 and CD62L markers and directly placed into ZymoResearch M-Digestion Buffer (2x) (cat# D5021-9) supplemented with proteinase K. Lysates were incubated at 65°C for >2 hours and then frozen. Bisulfite conversion and pyrosequencing of samples were performed by EpigenDx (assay ID ADS783-FS2) to examine the methylation status of nine CpG sites in intron 1 of the FOXP3 gene, spanning from the ATG to -2330 to -2263.

[0086] Heterozygous / homozygous integration prediction model If two HDR templates incorporating different fluorescent proteins at the same site are introduced (electroporated) into cells, an estimate of the proportion of cells carrying biallelic insertions (two potential alleles) at a single autosomal genomic locus can be made from the fluorescent phenotype alone. A simple probability model requires only two assumptions: Assumption 1: There are no off-target integrations at sites other than the target locus that contribute to the fluorescent phenotype. Assumption 2: Incorporation of a particular second fluorescent protein (i.e., RFP) is independent of which fluorescent protein has been incorporated into the other allele of the cell (i.e., GFP or RFP incorporation in the first allele is equally likely to result in RFP incorporation in the second allele).

[0087] After labeling in Figures 26A-C, the proportions of the four different phenotypic populations can be seen: ●% GFP - RFP - ●% GFP + RFP - ●% GFP - RFP + ●% GFP + RFP+

[0088] From this, two genotypes are immediately apparent: 1) Genotype A = NA / NA = % GFP - RFP - 2) Genotype E=GFP / RFP=% GFP + RFP +

[0089] The remaining four genotypes, taken together, reveal the two remaining single fluoro-positive phenotypes: 3) Genotype B + Genotype D = GFP / NA + GFP / GFP = % GFP + RFP - 4) Genotype C+genotype F=RFP / NA+RFP / RFP=% GFP - RFP +

[0090] Also, RFP + Cells express GFP + and GFP + Cells express RFP + The probability that there is a phenotype is: 5) RFP + GFP as + Probability that there is also a + RFP + ) / (% RFP + +% GFP + RFP + ) 6) GFP + RFP as + Probability that RFP|GFP is also true = P(RFP|GFP) = (% GFP + RFP + ) / (% GFP + +% GFP + RFP + )

[0091] According to assumption 2, if the probability that a cell undergoes GFP integration in its second allele is independent of whether the first integration was GFP or RFP, then the relationship between single-positive genotypes can be determined (Figure 26): 7) D = P(GFP|RFP)*B 8) F = P(RFP|GFP)*C

[0092] By inserting equations 7 and 8 into equations 3 and 4, respectively, and simplifying, the remaining genotype can be solved in terms of the known phenotype. 9) B = % GFP + RFP - / (1+(% GFP + RFP + ) / (% RFP + +% GFP + RFP + )) 10) C = % GFP - RFP + / (1+(% GFP + RFP + ) / (% GFP + +% GFP + RFP + )) 11) D = % GFP + RFP - -B 12) F = % GFP - RFP + -C

[0093] From a known genotype, the actual percentage of cells carrying monoallelic or biallelic insertions and other statistics can be easily calculated. Actual % of heterozygous cells = B + C Actual % of homozygous cells = D + E + F Actual % of cells with at least one insertion = B + C + D + E + F = 1 - A = 1 - % GFP - RFP - Actual % of alleles with GFP = (B + E + 2D) / 2 Measured % of alleles with RNP = (C + E + 2F) / 2 Actual % of alleles with insertions = % alleles GFP +% alleles RFP

[0094] If the HDR alleles are randomly distributed (essentially in Hardy-Weinberg equilibrium), the expected % of homozygous cells can be calculated from the observed % of cells with at least one insertion (HDR): ●p = HDR allele (GFP or RFP) ●q = non-HDR allele (NA) ●X = % of cells identified as having at least one HDR 13) p+q=1 14)p 2 +2*p*q+q 2 =1

[0095] Any cell carrying the HDR (GFP or RFP) allele will exhibit a phenotype (in this case, GFP+ or RFP+): 15) X=p 2 +2*p*q

[0096] Substituting X into equation 14 and simplifying, we get: 16)q=(1-X) 1 / 2 17) p=1-q 18) p = 1 - (1 - X) 1 / 2 p 2 gives the expected % of cells that are homozygous for HDR integration if HDR template insertion is random between the targeted alleles: 19)p 2 =2-2(1-X) 1 / 2 -X

[0097] Since X is known, the expected % homozygous cells can be calculated directly from the observed total % of cells with at least one HDR and then compared to the observed % homozygous cells calculated taking into account the information provided by the incorporation of two separate fluorophores.

[0098] Family history of autoimmune / immune dysregulation The proband was a Caucasian infant who, at 15 weeks of age, presented with vomiting, irritability, and tachypnea, leading to a medical evaluation revealing severe diabetic ketoacidosis and a serum glucose level of 920 mg / dL. One week after diagnosis, tests for GAD65, IA-2, and insulin autoantibodies were negative. However, at 5 to 7 months of age, autoimmune diabetes was confirmed when repeated antibody tests in three different laboratories showed positive results for IA-2 and insulin autoantibodies and very high levels of GAD65 antibodies in two of those laboratories [42.8 nmol / L (<0.02) at Mayo Laboratories and 896 IU / mL (0.0-5.0) at the Barbara Davis Center]. Tests for thyroid dysfunction and celiac disease were negative, but a slightly low IgA level suggested partial IgA deficiency. C-peptide testing was repeatedly completely undetectable, including a serum glucose level of 202 mg / dL measured 90 minutes after a meal at 7 months of age (proinsulin was also undetectable at that time). After initial DKA was treated with intravenous insulin, the infant was initially discharged and started on multiple daily injections of subcutaneous insulin (glargine and lispro), then transitioned to an insulin pump with continuous glucose monitoring. The infant consistently required high insulin replacement doses (48% basal at 7 months of age) ranging from 0.8 to 0.9 units / kg / day. The infant was delivered by repeat cesarean section at 37 weeks' gestation with a birth weight of 3.629 kg (75th percentile) and no complications, no concerns about growth progression, or an otherwise unremarkable medical history. The parents were of different Caucasian ancestry and were not consanguineous.

[0099] Clinical information for family members is provided in Table 1. Further details are as follows: 1. Mother (37): a. Pneumonia in childhood - explained as viral b. Treating ear infections with antibiotics as a child c. Dental problems (possibly antibiotic-related) d. My father developed insulin-dependent diabetes mellitus in his 30s. He had a low WBC count and nummular eczema on his scalp. e. Her mother had lupus 2. Father (44) Moroccan b. No particular medical problems c. A possible concern is that the response time to common viral infections may be long. 3. Affected children (14) a. Immune thrombocytopenic purpura: (+ antiplatelet antibodies) b. Neutropenia (anti-neutrophil antibodies) c. Autoimmune hemolytic anemia (DAT+, i.e., direct Coombs+) d. Nummular eczema on the scalp e. Hypercellular bone marrow: CD4 / CD8 ratio is inverted (0.36). f. stomatitis g. Treating ear infections with tubes h. Diarrhea in childhood i.46XX - No known chromosomal abnormalities j. Peripheral blood flow cytometry: 82.7% of CD45+ cells are CD3+ and 5.9% are CD19+. CD19+CD5+ cells are deficient B cells. 43.6% of CD45+ cells are CD8+, with an inverted CD4 / CD8 ratio (0.6). There is a relative increase in TCR(alphabeta)+CD3+CD4-CD8- T lymphocytes (26% TCRalphabeta+CD3+ cells, 5% CD45+ leukocytes). K. Has been treated with immunosuppressants including prednisone (20 mg), IgG-pro-IgA, Flonase nasal spray, and topical steroids and Symbicort. Also treated with Neupogen. 4. Affected children a.3+ diabetic autoantibodies (anti-GAD, MIAA, ICA, negative ZnT8 and ICA512 / IA-2) normal OGTT b. Treating an ear infection with a tube at age 1 C. Winter eczema 5. Unaffected daughter (15) a. Allergies, otherwise healthy 6. Affected son (4) a. Eczema in winter B. Positive HSV test c. Insulin-dependent diabetes mellitus (IDD) within the first year of life, C-peptide <0.1 at onset, anti-GAD antibody >30 (nl <1U / ml) at one year after diagnosis but negative at the time of diagnosis, ICA512 antibody 1.3 (nl <1.0) at one year after diagnosis but negative at the time of diagnosis 7. Unaffected daughter (9) a. Asthma

[0100] Genetic testing to identify IL2RA mutations Initial genetic testing of the proband using an in-house targeted next-generation sequencing multigene panel of over 40 genes known to be involved in monogenic forms of diabetes was negative. Subsequent exome sequencing in the proband and three of her parents revealed causative compound heterozygous mutations in the IL2RA gene. Two littermates carried only one mutation, while two others, carrying both mutations, had evidence of autoimmunity: the older brother was found to be diabetic autoantibody-positive in the absence of hyperglycemia (at age 4 or 5 years), and the older sister was diagnosed with autoimmune-mediated pancytopenia at age 11 years. CD25 expression was significantly reduced in the three compound heterozygotes.

[0101] Clinical phenotyping of IL2RA patients CD25-deficient children almost completely lose IL2-RA cell surface expression on T cells and therefore have virtually no detectable CD3+CD4+CD25hiCD127lo Tregs in their blood, whereas relatives carrying heterozygous IL2RA mutations show reduced CD25 expression on Tregs (Figure 34). However, the frequency of CD3+CD4+CD127loFOXP3+ T cells in CD25-deficient subjects is similar to that in HD and CD25+ / - individuals, suggesting that Tregs can develop in the absence of IL2-Ra function (Figure 34). Using a strategy to isolate Tregs without CD25 expression, we found that CD3+CD4+CD127loCD45RO+TIGIT+ Treg-enriched cells from CD25-deficient subjects exhibited a lack of ability to suppress the proliferation of responder T cells (Tresp) compared with their HD counterparts (Figure 34). In contrast, Tregs from relatives with a single heterozygous IL2RA mutation were able to suppress Tresp proliferation, albeit with suboptimal potency (Figure 34). Therefore, correcting functional IL2-Ra expression on the surface of FOXP3+ T cells from these patients may be a valuable approach for developing ex vivo gene therapy.

[0102] result Human T cells can be purified from blood, engineered ex vivo, and then reintroduced into the circulation by autologous transplantation. Engineered T cells have been developed to treat cancer and infectious diseases (Fesnak et al. "Engineered T cells: the promise and challenges of cancer immunotherapy," Nat. Rev. Cancer 16, 566-581 (2016); and Esensten et al. "Engineering Therapeutic T Cells: From Synthetic Biology to Clinical Trials," Annu. Rev. Pathol. 12, 305-330 (2017)).

[0103] These cell-based therapies rely on the ability to genetically reprogram T cells, for example, to enhance their ability to recognize and attack specific antigens (Roybal et al. "Synthetic Immunology: Hacking Immune Cells to Expand Their Therapeutic Capabilities," Annu. Rev. Immunol. 35, 229-253 (2017)). Cell-based therapies, including engineered regulatory T cells (Tregs), designed to suppress inflammation, are being developed for autoimmune diseases and organ transplants (Bluestone et al. "Type 1 diabetes immunotherapy using polyclonal regulatory T cells," Sci. Transl. Med. 7, 315ra189 (2015)).

[0104] Various approaches have been used to modify the genomes of primary human T cells. Lentiviral vectors can be used to insert long DNA sequences (multiple kilobases), but the integration site is non-targeted (Verhoeyen et al. in Methods in Molecular Biology (2009), pp. 97-114). Lentiviruses have been the primary means for introducing gene constructs such as chimeric antigen receptors (CARs) (Kalos et al., "T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia," Sci. Transl. Med. 3, 95ra73 (2011)). To knock out specific endogenous genes, sequence-specific nucleases such as Cas9, TALEN, or zinc finger nucleases (ZFNs) can be electroporated into T cells (Schumann et al., "Generation of knock-in primary human T cells using Cas9 ribo nucleoproteins," Proceedings of the National Academy of Sciences. 112, 10437-10442 (2015); and Perez et al. "Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases," Nat. Biotechnol. 26, 808-816 (2008)) to generate double-strand breaks that generate a nonrandom series of insertion and deletion mutations through nonhomologous end joining (NHEJ) (van Overbeek et al., "DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks," Mol. Cell. 63, 633-646 (2016)).Co-delivery of small (<200 bp) chemically synthesized ssDNA oligos (ssODNs) with homology to sequences flanking specific nuclease cleavage sites has been used to modify short DNA sequences by homology-directed repair ( Schumann et al. (2015) ).

[0105] Targeted integration of much longer DNA sequences would enable more diverse applications. This has recently been achieved by electroporation of sequence-specific nucleases followed by infection with an integrase-deficient adeno-associated vector (AAV) containing an HDR template (Sather et al., "Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template," Sci. Transl. Med. 7, 307ra156 (2015); and Hubbard et al. "Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome," Blood 127, 2513-2522 (2016)). This electroporation-infection method has enabled a novel therapeutic T cell engineering strategy (Eyquem et al., "Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection," Nature 543, 113-117 (2017)), but it is prone to off-target integration, requires potentially unwanted viral infection, and has limited throughput due to challenges in virus production.

[0106] To develop a highly efficient nonviral genome targeting method, we tested cell culture conditions, Cas9 RNP and HDR template concentrations, and electroporation parameters. We identified conditions that allowed for the co-delivery of high concentrations of Cas9 RNP and long DNA templates (>1 kb) into multiple loci in primary human T cells with minimal impact on cell viability.

[0107] Nonviral targeting can be used to correct pathogenic mutations that cause Treg dysfunction and monogenic autoimmune diseases. Herein, we describe a family in which two children developed early-onset autoimmune disease and the third had autoantibodies suggestive of a very high risk for type 1 diabetes (T1D), and a causative loss-of-function mutation in IL2RA was identified through exome sequencing. IL2RA is crucial for regulatory T cell function and immune homeostasis. The nonviral CRISPR genome targeting method provided herein achieved efficient mutation correction, restoring cell surface expression of IL2RA along with functional downstream signaling. Nonviral genome targeting in primary human immune cells allows for functional study and correction of mutations in cells from patients. Cell therapy combined with improved gene targeting (nonviral templates, high efficiency and specificity, and long targeting constructs) holds great promise for the treatment of autoimmune diseases as well as immunodeficiencies, infectious diseases, organ transplants, and cancer immunotherapy.

[0108] Development of non-viral human T cell genome targeting The main limitation of genome targeting in human T cells has been that DNA delivery leads to cell death (Cornu et al., "Refining strategies to translate genome editing to the clinic," Nat. Med. 23, 415-423 (2017)). Introduction of short single-stranded oligodeoxynucleotide (ssODN) HDR templates did not result in significant loss of T cell viability, whereas larger linear dsDNA templates resulted in extensive toxicity (Y. Zhao et al., "High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation," Mol. Ther. 13, 151-159 (2006); and Hornung et al. "Intracellular DNA Recognition," I10, 123-130 (2010)).

[0109] As shown herein, long (>1 kb) linear dsDNA templates exhibited lower toxicity when co-electroporated with CRISPR-Cas9 ribonucleoprotein (Cas9 RNP) (Figure 10). This suggested that co-delivery of an appropriate mixture of Cas9 RNP and long dsDNA enables HDR and preserves cell viability.

[0110] We optimized nonviral genome targeting in primary human T cells. We adjusted the protocol for targeted integration efficiency, cell viability, and total number of integration-positive cells (Figures 11A and 12). Cas9 RNP was electroporated with a dsDNA HDR template designed to introduce an N-terminal GFP fusion to the housekeeping gene RAB11A (Figure 11B). Integration and cell viability were monitored using high-throughput flow cytometry performed 3–5 days after electroporation. First, we identified stimulation and cytokine treatments that significantly increased the rate of gene targeting before and after electroporation (Figure 11C and Figures 13 and 14). These conditions enabled efficient targeting in fresh or frozen primary T cells isolated from various sources (Figure 15). We tested various concentration ratios of Cas9 RNP to HDR template in these well-stimulated T cells (Figure 11D and Figure 16) to identify the appropriate concentration that enabled efficient gene targeting. Finally, we tested electroporation conditions (Figure 11E and Figure 17) to maximize gene targeting while retaining high levels of cell viability. Nonviral gene targeting was achieved by introducing a GFP fusion to the endogenous RAB11A housekeeping gene into more than 50% of cells in both primary human CD4+ and CD8+ T cells (Figure 11F).

[0111] High-throughput combinatorial gene targeting applications The simplicity and speed of the methods provided herein for non-viral gene targeting across genomic sites and human blood donors (FIG. 18 and FIG. 12). Constructs encoding GFP fusions with homologous flanking sequences were efficiently and reproducibly targeted to diverse sites throughout the genome (FIG. 18A and FIG. 19). These targeted GFP fusions labeled diverse subcellular structures (Leonetti et al. "A scalable strategy for high-throughput GFP tagging of endogenous human proteins," Proc. Natl. Acad. Sci. USA 113, E3501-8 (2016)). Confocal microscopy confirmed the specificity of fusion proteins generated by targeting diverse genes and demonstrated that targeting endogenous genes with GFP allows imaging of protein localization in live human T cells (FIG. 18B). We demonstrated that targeted GFP integration into diverse genes was highly reproducible in primary human T cells from a large cohort of healthy donors (Figures 19 and 20). Furthermore, tagging the endogenously encoded CD4 surface receptor with GFP confirmed the specificity of targeted integration and the cell-type-specific expression pattern of the tagged gene. Specifically, we observed a linear relationship between CD4 and GFP expression in tagged CD4+ T cells but not in CD8+ T cells (Figure 18C). Together, these findings demonstrate that nonviral genome targeting can be used to modify endogenous genes by inserting large DNA sequences into targeted sites in the genome.

[0112] Fusion tags not only enabled imaging of endogenous proteins but could also be used for biochemical targeting of specific proteins. For example, ChIP-Seq and more recently CUT&RUN (Skene and Henikoff, "An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites," ELife 6 (2017), doi: 0.7554 / eLife.21856.) have been widely used to map transcription factor binding sites. However, these assays are often limited by the availability of effective and specific antibodies. As proof of principle, we performed CUT&RUN using an anti-GFP antibody in primary T cells in which the endogenous gene encoding BATF (a pivotal TF) had been targeted to generate a GFP fusion. The binding site identified by anti-GFP CUT&RUN closely matched that identified by anti-BATF (Figure 18D and Figure 21).

[0113] Targeting two alleles of the same gene with two different fluorophores would provide a method for quantifying and enriching cells with biallelic modifications. Introducing two different fluorescent proteins targeting the same site in the RAB11A gene (Figures 22A and 23) demonstrated successful biallelic integration in >5% of cells. Importantly, because some cells would have received either GFP or mCherry on both alleles, the number of cells expressing both fluorescent proteins underestimates the proportion of cells with biallelic integration. We constructed a model to account for homozygous integration of the same fluorescent protein (Figures 22B and 23). This model estimates that up to approximately 10% of cells had biallelic integration in the RAB11A gene. This suggests that cells with one RAB11A integration are more likely to have also received a second targeted integration; this effect was observed across three genomic loci (Figure 23). Simultaneous delivery of three fluorescent tags targeting the RAB11A locus demonstrated a very low percentage of cells expressing all three fluorophores, consistent with the low rate of off-target integration in these experiments (Figure 23G). In summary, the use of multiple non-viral constructs to target the same locus allows for the identification of biallelic genome editing in human T cells.

[0114] Multiplexed editing of combinatorial sets of genomic sites will provide expanded research and therapeutic applications. We tested whether co-delivery of multiple nonviral HDR templates with multiple RNPs could generate primary cells with more than one modified locus. Not only was multiplexed gene targeting possible (Figure 22C), but we also found that gating on cells with one modification enriched for cells with two modifications (Figure 22D and Figure 24) (Agudelo et al., "Marker-free coselection for CRISPR-driven genome editing in human cells," Nat. Methods. 14, 615-620 (2017)). Triple gene targeting was also achieved, where gating on cells with two targeted insertions significantly enriched for cells with a third modification (Figure 22E and Figure 24). Overall, nonviral gene targeting can be used to enable complex genetic modification of primary T cells, which can be used for diverse research and therapeutic applications.

[0115] D10A nickase and ssDNA HDR templates reduce off-target integration One of the major concerns with using HDR templates, especially for therapeutic applications, is the potential for off-target integration. This has also been observed when integrase-deficient AAV was used as the donor template (Dever et al., "CRISPR / Cas9 β-globin gene targeting in human hematopoietic stem cells," Nature 539, 384-389 (2016)). Similar evidence of functional off-target integration using linear dsDNA templates for non-viral gene targeting was found here. Double-stranded DNA templates can integrate into sites of spontaneous dsDNA breaks (Murnane et al. "Recombination events during integration of transfected DNA into normal human cells," Nucleic Acids Res. 18, 2733-2738 (1990)) as well as into specific dsDNA breaks induced by targeted nucleases such as Cas9 in an HDR-independent manner (an effect called homology-independent targeted integration) (Auer et al. "Highly efficient CRISPR / Cas9-mediated knock-in in zebrafish by homology-independent DNA repair," Genome Res. 24, 142-153 (2014); and Suzuki et al. "In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration," Nature 540, 144-149 (2016)). We searched for unintended non-homologous integration using an N-terminal GFP-RAB11A fusion construct containing the endogenous RAB11A promoter sequence within its 5' homology arm, which can drive GFP expression at off-target integration sites (Figures 25A and 26).Functional off-target integration was found to be present in cells from different biological donors (Figure 25B) and observed in experiments using various target sequences and HDR templates (Figures 26 and 27). In cells intended for therapeutic use, off-target integration must be minimized to ensure that the integrated sequence remains under the correct endogenous control and that off-target sites are not disrupted.

[0116] To reduce off-target integration caused by off-target Cas9 cleavage, non-viral gene targeting was performed using the D10A Cas9 nickase mutant. This mutant requires two gRNAs to bind and cleave in close proximity to form double-strand breaks, thereby reducing the number of off-target dsDNA breaks (Miyaoka et al., "Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing," Sci. Rep. 6 (2016), doi:10.1038 / srep23549; Vriend et al., "Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks, and paired nicks," Nucleic Acids Res. 44, 5204-5217 (2016); and Bothmer et al., "Characterization of the interplay between DNA repair and CRISPR / Cas9-induced DNA lesions at an endogenous locus," Nat. Commun. 8, 13905 (2017)). A series of gRNA combinations were tested at the RAB11A locus for GFP integration, and a set of "PAM-Out" guides was found that showed efficient introduction of GFP when using D10A nickase (Figure 28). As expected, the use of D10A with a single off-target guide showed significantly reduced functional off-target integration compared to Cas9, equivalent to the level seen when using nuclease-incompetent dCas9 (Figure 25C).

[0117] Even with D10A nickase, the dsDNA HDR template still resulted in rare but observable off-target integration (comparable to rates observed without Cas9 nuclease), presumably at spontaneous dsDNA breaks (Figure 25A and C). We reasoned that by replacing the dsDNA HDR template with a long ssDNA HDR template that cannot nonspecifically integrate at double-strand breaks, we could eliminate the remaining off-target integration (Quadros et al., "Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins," Genome Biol. 18, 92 (2017); and Leonetti et al. http: / / www.biorxiv.org / content / early / 2017 / 08 / 21 / 178905).

[0118] To test this hypothesis, we generated ssDNA HDR templates by two methods that generate the large amounts of long ssDNA required for electroporation (Figure 35). The ssDNA HDR template reduced functional off-target integration by approximately 100-fold while maintaining efficient on-target integration (Figure 25D). It was possible to use D10A Cas9 nickase with the ssDNA template. In these experiments, the on-target integration rate was reduced, while nonspecific integration was reduced to background levels seen without the template (Figures 25E and F). For sites where potential off-target activity is a concern, D10A Cas9 nickase and the ssDNA HDR template can be used to reduce the rate of integration resulting from off-target-induced double-strand breaks and spontaneous breaks, respectively, making this method attractive for therapeutic modification of patient T cells.

[0119] Therapeutic mutation correction by non-viral gene targeting The application of non-viral gene targeting to correct mutations causing monogenic immune dysregulation in T cells from patients was explored. We have identified families with monogenic primary immune dysregulation with autoimmune disease caused by recessive loss-of-function mutations in the gene encoding the IL-2 alpha receptor (IL2RA), also known as CD25 (Sharfe et al. "Human immune disorder arising from mutation of the alpha chain of the interleukin-2 receptor," Proc. Natl. Acad. Sci. USA 94, 3168-3171 (1997); Goudy et al. "CD25 deficiency causes an immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome, and defective IL-10 expression from CD4 lymphocytes," J. Allergy Clin. Immunol. 119, 482-487 (2007); and Goudy et al., "Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity," Clin. Immunol. 146, 248-261 (2013).IL2RA is essential for Tregs and immune homeostasis (Sakaguchi et al. "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases," J. Immunol. 155, 1151-1164 (1995); and Rudensky et al. "Regulatory T cells and Foxp3," Immunol. Rev. 241, 260-268 (2011)). Children from a compound heterozygous family carrying two loss-of-function mutations exhibit pleiotropic autoimmune manifestations (Table 1). One child developed neonatal-onset type 1 diabetes (T1D), and the other developed refractory autoimmune cytopenias during childhood. All three IL2RA-deficient family members exhibited pathological serum autoantibodies. IL2RA-deficient children almost completely lose IL2RA cell surface expression, and thus CD3+CD4+CD25hiCD127lo Tregs are virtually undetectable in the blood, whereas relatives with heterozygous IL2RA mutations show reduced IL2RA expression on Tregs (Figure 30). However, the frequency of CD3+CD4+CD1271oFOXP3+ T cells in IL2RA-deficient subjects is similar to that in healthy donors (HD) and heterozygous family members, suggesting that Treg-like cells develop and persist regardless of IL2RA mutation. Using a strategy to isolate Tregs without CD25 expression, we found that CD3+CD4+CD1271oCD45RO+TIGIT+ Treg-enriched cells from CD25-deficient subjects exhibited a lack of ability to suppress responder T cell (Tresp) proliferation compared to their HD counterparts (Figure 29). In contrast, Tregs from relatives with a single heterozygous IL2RA mutation were able to suppress Tresp proliferation, albeit with suboptimal potency (FIG. 29).Therefore, correcting functional IL2RA expression on the surface of FOXP3+ T cells from these patients may be a valuable approach for developing ex vivo gene therapy.

[0120] [Table 1]

[0121] Whole-exome sequencing revealed that the IL2RA-deficient child harbored a compound heterozygous mutation in IL2RA (Figure 30A and Figure 31). One mutation, c.530A>G, creates a premature stop codon. Improvements in cell culture and electroporation methodology allowed for efficient correction of the mutation using a chemically synthesized ssDNA HDR template of approximately 120 bp (Figure 32). The rate was even higher when longer dsDNA templates were used (Figure 30B and Figures 32 and 33). Corrected patient-derived T cells expressed IL2RA on their surface. Correction was successful in all three litters, but a lower IL2RA expression rate was observed in compound het 3, which may be due to an altered cellular state associated with the patient's disease or the fact that the patient was the only litter member treated with immunosuppressive drugs (Table 1 and Figure 34). The second mutation, c.800delA, causes a frameshift in the reading frame of the final IL2RA exon, resulting in misreading of the portion of the gene encoded by the final exon and run-on translation past the normal stop codon. This frameshift can be corrected even without an HDR template (Figure 33). At this site, genomic cleavage produced by Cas9 RNP alone was sufficient to allow productive cell surface expression of IL2RA, presumably by restoring the correct frame through an insertion / deletion mutation (Figure 33). Together, these data demonstrate how different mutations in patient T cells can be corrected using HDR template-dependent and non-HDR template-dependent repair mechanisms.

[0122] One possible therapeutic strategy for this family member with a monogenic Treg deficiency would be ex vivo T cell gene correction followed by infusion of corrected autologous Tregs. Treg cells generated by targeted correction could mitigate some of the potential risks of hematopoietic stem cell transplantation. We tested whether correcting one of the IL2RA mutations would lead to productive signaling and whether correction would occur in a significant proportion of FOXP3+ Tregs. After correction of the c.530A>G mutation, the cells were able to functionally signal through IL2RA, the high-affinity IL-2 receptor. In response to IL-2 treatment, the modified cells showed increased STAT5 phosphorylation, indicative of productive signaling (Figure 31C and Figures 33 and 34). In addition, flow cytometry confirmed that a portion of the IL2RA-corrected cells expressed FOXP3, a critical transcription factor for Tregs (Figure 30D and Figures 32 and 33).

[0123] The endogenous gene encoding IL2RA is under strict control by multiple cis-regulatory elements that constitute a super-enhancer (Farh et al., "Genetic and epigenetic fine mapping of causal autoimmune disease variants," Nature 518, 337-343 (2015); and Simeonov et al. "Discovery of stimulation-responsive immune enhancers with unbiased CRISPR activation," Nature 549 (7670): 111-115 (2017)). Therefore, therapeutic correction of IL2RA likely relies on specific repair of the gene in its endogenous genomic locus. Given that GFP insertion using Cas9 and dsDNA demonstrated the possibility of nonspecific integration of dsDNA, we specifically repaired the c.530A>G patient mutation using D10A Cas9 nickase and a long ssDNA template. Using these reagents, we were able to specifically and selectively correct the mutant gene in approximately 20% of T cells from the patient (Figure 30E).

[0124] Non-viral gene targeting allows efficient insertion of defined sequences throughout the genome of primary human T cells. These insertions can range from the introduction or correction of single base pair mutations to the integration of large functional sequences and tags in endogenous loci, allowing for multiplexed integration throughout the genome. For the therapeutic use of engineered T cells, the use of D10A Cas9 nickase and ssDNA HDR template can significantly reduce off-target integration. The methods and results provided herein enable the accelerated development of engineered T cell therapy and the treatment of genetic diseases.

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[Claim 1] The invention described herein.

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