Optimized crispr / cas9 systems and methods for gene editing in stem cells

Abstract

To provide methods and compositions that can be used to optimize gene editing or regulation in stem cells to preserve the viability, multipotency, and self-renewal capability of stem cells.SOLUTION: The methods and compositions described herein surprisingly increase CRISPR / Cas-mediated gene editing in stem cells by transiently treating the cells with a stem cell viability enhancer prior to and / or after contacting the cells with one or more CRISPR / Cas9 components. Further, this treatment also surprisingly results in increased engraftment of the stem cells into the target tissue of a subject. The present disclosure also provides one or more modified CRISPR / Cas9 components which, when used in combination with the stem cell viability enhancer, further increases the frequency of gene editing in stem cells, increases stem cell viability, and increases stem cell engraftment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 159,785, filed March 11, 2015; U.S. Provisional Patent Application No. 62 / 220,648, filed September 18, 2015; U.S. Provisional Patent Application No. 62 / 244,577, filed October 21, 2015; and U.S. Provisional Patent Application No. 62 / 279,020, filed January 15, 2016, the entire contents of each of which are expressly incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on May 6, 2016, is named 2016-05-06_126454-00520_ST25.txt, and is 1.07 megabytes in size. [Background technology]

[0003] Gene therapy is a series of strategies used to modify the expression of genes in an individual or correct abnormal genes. Cell therapy involves administering viable cells to a patient undergoing disease treatment or maturing a specific cell population in the patient. Gene therapy and cell therapy are common fields that aim to target the cause of disease in nucleic acids or cell populations. For example, hematopoietic diseases can be treated by transplanting ex vivo gene-modified stem cells (e.g., hematopoietic stem / progenitor cells and hematopoietic stem cells, also known as HSCs) into a subject.

[0004] The discovery of the CRISPR / Cas9 system and its application to mammals has enabled efficient and precise editing of target genes, for example, via non-homologous end joining (NHEJ), homology-directed repair (HDR), or other DNA repair pathways. Co-delivery of Cas9 molecules and target-specific guide RNA (gRNA) molecules, optionally along with donor DNA repair template molecules, facilitates gene editing of target sequences (e.g., disease-associated mutations) within the genome. Therefore, the use of the CRISPR / Cas9 system to modify genes in stem cells is a promising strategy for treating multiple genetic diseases. However, because stem cells are highly sensitive to manipulation in vitro and ex vivo, manipulation of stem cells with the CRISPR / Cas9 system has been inefficient to date, with few successful attempts to achieve long-term stem cell viability and engraftment in vivo.

[0005] To facilitate the use of stem cells (e.g., HSCs), methods for expanding stem cells in vitro have been developed. For example, certain small molecules have been used to expand stem cells in vitro, e.g., to increase proliferation, i.e., to increase the number of stem cells in culture by several times, over a long exposure period (typically more than one week). In such cases, a minimum exposure period of at least 7 days is required to promote stem cell expansion, e.g., to significantly increase the number of stem cells in culture in vitro. However, this long in vitro culture period required to expand stem cell populations is not optimal for clinical application.

[0006] Furthermore, the clinical efficacy of cell transplants using expanded stem cells is often contingent on achieving a threshold level of engraftment, which to date has been extremely difficult to achieve after stem cell manipulation with the CRISPR / Cas9 system. See, e.g., Walasek et al., Ann. N.Y. Acad. Sci. (2012), 1266:138-150. In fact, to date, no published reports have demonstrated long-term engraftment of more than 1% of HSCs manipulated with any CRISPR / Cas9 system, even after expansion. (Mandal et al. (2014) Cell Stem Cell 15(5):643-52) [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Walasek et al.,Ann.NYAcad.Sci.(2012),1266:138-150 [Non-patent document 2] Mandal et al.(2014)Cell Stem Cell 15(5):643-52 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need for alternative methods and compositions that can be used to optimize gene editing or regulation in stem cells (e.g., HSCs) to preserve the viability, multipotency, and self-renewal capacity of stem cells. [Means for solving the problem]

[0009] The methods and compositions described herein enhance CRISPR / Cas-mediated gene editing in stem cells, such as HSCs. Exposing stem cells to foreign molecules, such as CRISPR / Cas9 system components (e.g., gRNA molecules, Cas9 molecules, or template nucleic acids), can stress stem cells and induce innate immune responses, which can trigger, for example, programmed cell death or stem cell differentiation. Using the methods and systems described herein, the innate immune response signaling events that can ultimately lead to programmed cell death of stem cells after exposure to foreign CRISPR / Cas9 components are reduced or neutralized. Specifically, by temporarily (e.g., for a period of less than 120 hours) exposing stem cells to a stem cell viability enhancer before and / or after contacting them with one or more CRISPR / Cas9 components, the innate immune response of stem cells to foreign CRISPR / Cas components is reduced or inhibited, thereby significantly increasing stem cell viability.

[0010] Furthermore, transient treatment of stem cells with stem cell viability enhancers (e.g., small molecules) that improve viability, inhibit intracellular innate immune responses, or both, before and / or after delivery of CRISPR / Cas9 components also results in increased stem cell multipotency and self-renewal capacity. Furthermore, transient treatment of stem cells with stem cell viability enhancers (e.g., small molecules) that improve viability, inhibit intracellular innate immune responses, or both, before and / or after delivery of CRISPR / Cas9 components also increases stem cell engraftment in target tissues upon introduction of the modified stem cells into a subject. The unexpected benefit of transiently exposing stem cells to the reagents disclosed herein is surprising, considering that these are typically used as stem cell expansion agents, e.g., to increase stem cell proliferation and increase their number several-fold, and the benefits of stem cell expansion require prolonged contact with the agent (e.g., exposure in culture for more than one week). Thus, the methods and compositions described herein are particularly advantageous for optimizing the editing of target nucleic acid sequences in viable stem cells and advancing the field of stem cell therapy in a variety of clinical applications.

[0011] In one aspect, disclosed herein is a method of producing modified cells, e.g., stem cells, for transplantation, comprising: (a) contacting cells, e.g., stem cells, with a stem cell viability enhancer for a period of less than 120 hours, followed by (b) contacting the cells with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer.

[0012] In another aspect, disclosed herein is a method for modifying a target nucleic acid in a cell, e.g., a stem cell, comprising contacting the cell with a stem cell viability enhancer, a modified gRNA molecule, and a Cas9 molecule. In one embodiment, the contacting comprises (a) contacting the cell with the stem cell viability enhancer for a period of less than 120 hours, followed by (b) contacting the cell with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer.

[0013] In another aspect, disclosed herein is a method for transplanting modified cells, e.g., stem cells, into a subject, the method comprising contacting cells, e.g., stem cells, with a stem cell viability enhancer; a gRNA molecule; and a Cas9 molecule to generate modified cells, e.g., modified stem cells, and transferring the modified cells, e.g., modified stem cells, into the subject. In one embodiment, the contacting comprises (a) contacting the cells, e.g., stem cells, with the stem cell viability enhancer for a period of less than 120 hours, followed by (b) contacting the cells, e.g., stem cells, with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer.

[0014] In one embodiment, the step of contacting the cell, e.g., stem cell, with the gRNA molecule and Cas9 molecule is performed using electroporation.

[0015] In one embodiment, the method further comprises subjecting the cells, e.g., stem cells, to a cold shock prior to electroporation. In one embodiment, the method further comprises subjecting the cells, e.g., stem cells, to a cold shock after electroporation. In one embodiment, the cells, e.g., stem cells, are subjected to a cold shock at a temperature of about 30°C to about 32°C.

[0016] In another embodiment, the period of less than 120 hours is about 96 hours. In one embodiment, the period of less than 120 hours is about 72 hours. In another embodiment, the period of less than 120 hours is about 48 hours. In another embodiment, the period of less than 120 hours is about 36 hours. In another embodiment, the period of less than 120 hours is about 24 hours. In another embodiment, the period of less than 120 hours is about 12 hours. In another embodiment, the period of less than 120 hours is about 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 hours. In another embodiment, the period of less than 120 hours is about 24 to about 48 hours. In another embodiment, the period of less than 120 hours is about 1 to about 120 hours. In another embodiment, the period of less than 120 hours is about 48 to about 120 hours. In another embodiment, the period of less than 120 hours is about 24 to about 96 hours. In another embodiment, the period of less than 120 hours is about 24 to about 72 hours. In another embodiment, the period of less than 120 hours is about 36 to about 48 hours. In another embodiment, the period of less than 120 hours is about 24 to about 36 hours. In another embodiment, the period of less than 120 hours is about 12 to about 24 hours. In another embodiment, the period of less than 120 hours is about 12 to about 36 hours. In one embodiment, a period of less than 120 hours is not long enough to promote expansion of stem cells.

[0017] In one embodiment, the method further comprises (c) contacting the cells, e.g., stem cells, with the stem cell viability enhancer for a period of less than 96 hours after step (b). In one embodiment, the method further comprises (c) contacting the cells, e.g., stem cells, with the stem cell viability enhancer for a period of less than 84 hours after step (b).

[0018] In one embodiment, the method further comprises (c) contacting the cells, e.g., stem cells, with a stem cell viability enhancer for a period of less than 72 hours after step (b). In one embodiment, the method further comprises contacting the cells, e.g., stem cells, with a stem cell viability enhancer for a period of less than 72 hours after step (b). In one embodiment, the period of less than 72 hours after step (b) is not long enough to result in expansion of the cells.

[0019] In one embodiment, the cells are a population of cells, and the number of cells in the population of cells does not increase by more than 10-fold in less than 72 hours after step (b). In one embodiment, the cells are a population of cells, and the number of cells in the population of cells does not increase by more than 5-fold in less than 72 hours after step (b). In another embodiment, the cells are a population of cells, and the number of cells in the population of cells does not increase by more than 4-fold in less than 72 hours after step (b). In another embodiment, the cells are a population of cells, and the number of cells in the population of cells does not increase by more than 3-fold in less than 72 hours after step (b). In another embodiment, the cells are a population of cells, and the number of cells in the population of cells does not increase by more than 2-fold in less than 72 hours after step (b).

[0020] In one embodiment, the period of less than 72 hours after step (b) is a period of about 24 to about 48 hours after step (b). In one embodiment, the period of less than 72 hours after step (b) is about 12 hours, 24 hours, 36 hours, 48 ​​hours, or 60 hours after step (b).

[0021] In one embodiment, the cells, e.g., stem cells, are transferred into the human subject within 96 hours of completion of one or more contacting steps. In one embodiment, the stem cells are transferred into the human subject within 72 hours of completion of one or more contacting steps. In one embodiment, the stem cells are transferred into the human subject within 60 hours of completion of one or more contacting steps. In one embodiment, the stem cells are transferred into the human subject within 48 hours of completion of one or more contacting steps. In one embodiment, the stem cells are transferred into the human subject within 36 hours of completion of one or more contacting steps. In one embodiment, the stem cells are transferred into the human subject within 24 hours of completion of one or more contacting steps.

[0022] In one embodiment, the cells, e.g., stem cells, are cryopreserved within 96 hours of completion of one or more contacting steps. In one embodiment, the stem cells are cryopreserved within 72 hours of completion of one or more contacting steps. In one embodiment, the stem cells are cryopreserved within 60 hours of completion of one or more contacting steps. In one embodiment, the stem cells are cryopreserved within 48 hours of completion of one or more contacting steps. In one embodiment, the stem cells are cryopreserved within 36 hours of completion of one or more contacting steps. In one embodiment, the stem cells are cryopreserved within 24 hours of completion of one or more contacting steps.

[0023] In one embodiment, the stem cell viability enhancer inhibits differentiation of stem cells. In another embodiment, the stem cell viability enhancer inhibits programmed cell death of stem cells. In another embodiment, the stem cell viability enhancer inhibits senescence of stem cells. In another embodiment, the stem cell viability enhancer inhibits the innate immune response of stem cells. In one embodiment, the stem cell viability enhancer inhibits programmed cell death of stem cells by inhibiting autophagy or apoptosis of stem cells.

[0024] In one embodiment, the stem cells engraft into the target tissue of the subject.

[0025] In one embodiment, the stem cells comprise a population of stem cells, wherein at least 2% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 3% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 4% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 5% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 6%, 7%, 8%, 9%, or 10% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 15% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 20% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 25% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cells comprise a population of stem cells, wherein at least 30%, 35%, 40%, 45%, or 50% of the stem cells engraft into the target tissue of the subject.

[0026] In one embodiment, the cells comprise a population of cells, wherein at least 1% of the cells are capable of engrafting into the subject's bone marrow. In another embodiment, at least 5% of the cells are capable of engrafting into the subject's bone marrow. In another embodiment, at least 10% of the cells are capable of engrafting into the subject's bone marrow. In another embodiment, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the cells are capable of engrafting into the subject's bone marrow.

[0027] In one embodiment, the cells comprise a population of cells wherein at least 1% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 2.5% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 5% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 10% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 20% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 25% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 30% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 35% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 40% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 45% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 50% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 55% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 60% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 65% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 70% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 75% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 80% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 85% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 80% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 85% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 90% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 95% of the cells reconstitute the subject's peripheral blood. In another embodiment, at least 100% of the cells reconstitute the subject's peripheral blood. In one embodiment, the stem cells remain engrafted in the subject's target tissue for at least 16 weeks. In another embodiment, the stem cells remain engrafted in the subject's target tissue for at least 20 weeks. In another embodiment, the stem cells remain engrafted in the subject's target tissue for at least 24 weeks. In another embodiment, the stem cells remain engrafted in the target tissue of the subject for at least six months.In another embodiment, stem cells remain engrafted in the target tissue of the subject for at least 9 months.In another embodiment, stem cells remain engrafted in the target tissue of the subject for at least 1 year.In another embodiment, cells can be engrafted in the target tissue of the subject for the rest of the subject's life.

[0028] In one embodiment, the target tissue is peripheral blood, bone marrow, or spleen. In one embodiment, the cells are stem cells. In one embodiment, the stem cells are hematopoietic stem / progenitor cells (HSCs). As used herein, the term HSCs refers to both hematopoietic stem cells and hematopoietic stem / progenitor cells. In one embodiment, the stem cells are selected from the group consisting of circulating blood cells, mobilized blood cells, bone marrow cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, lineage-specific progenitor cells, endothelial cells, or mesenchymal stromal cells. In another embodiment, the HSCs are non-cord blood-derived, umbilical cord-derived, or cord blood-derived. In one embodiment, the HSCs are CD34+ cells.

[0029] In one embodiment, the method further comprises isolating cells, eg, stem cells, from the subject prior to the one or more contacting steps.

[0030] In one embodiment, the method further comprises, after step (b), culturing the cells, e.g., stem cells, in a medium containing one or more cytokines. In one embodiment, the medium comprises one or more cytokines and a stem cell viability enhancer. In one embodiment, the one or more cytokines are selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

[0031] In one embodiment, the method further comprises, after step (b), culturing the cells, e.g., stem cells, in a medium comprising one or more of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-β signaling modulator, insulin-like growth factor binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), angiopoietin (ANG1), angiopoietin-like protein (ANGPTL4), an SDF1 / CXCR4 transduction axis modulator, a Wnt signaling modulator, or a combination thereof.

[0032] In one embodiment, the cells, eg, stem cells, are cultured in the medium for at least 1, 2, 3, 4, 5, 6, or 7 days.

[0033] In one embodiment, the stem cell viability enhancer is an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist. In one embodiment, the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), LGC0006, α-naphthoflavone, and CH-223191. In one embodiment, the AhR antagonist is SR1. In one embodiment, the innate immune response antagonist is selected from the group consisting of cyclosporin A, dexamethasone, reservatrol, MyD88 inhibitory peptides, RNAi agents targeting Myd88, B18R recombinant protein, glucocorticoids, OxPAPC, TLR antagonists, rapamycin, BX795, and RLR shRNA. In one embodiment, the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16,16-dimethylprostaglandin E2 (dmPGE2).

[0034] In one embodiment, the Cas9 molecule is enzymatically active Cas9 (eaCas9). In one embodiment, the Cas9 molecule is selected from the group consisting of wild-type Cas9, nickase Cas9, inactive Cas9 (dCas9), split Cas9, and inducible Cas9. In one embodiment, the Cas9 molecule comprises N-terminal RuvC-like domain cleavage activity but lacks HNH-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position N863 of Streptococcus pyogenes Cas9. In one embodiment, the Cas9 molecule comprises HNH-like domain cleavage activity but lacks N-terminal RuvC-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

[0035] In one embodiment, the Cas9 molecule is a Cas9 polypeptide. In one embodiment, the gRNA molecule and the Cas9 polypeptide are combined in a preformed ribonucleotide complex. In one embodiment, the Cas9 molecule is a nucleic acid encoding the Cas9 polypeptide.

[0036] In one embodiment, the gRNA molecule comprises a 5'-end cap structure. In one embodiment, the gRNA molecule comprises a 3'-end poly-A tail.

[0037] In one embodiment, the method further comprises contacting cells with template nucleic acid.In one embodiment, the cell is contacted with template nucleic acid during the same contacting step as gRNA molecule and Cas9 molecule.In one embodiment, the template nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).In one embodiment, the ssODN comprises 5' phosphorothioate modification, 3' phosphorothioate modification, or a combination thereof.

[0038] In one embodiment, the method further comprises contacting the stem cell with a transgene, wherein the contacting step is performed under conditions that allow the transgene to be integrated into the genome of the stem cell. In one embodiment, the transgene is a gene that is a chemotherapy selectable marker, a cell surface antigen, or a suicide gene.

[0039] In one embodiment, the transgene is integrated into a safe harbor locus. In one embodiment, the safe harbor locus is the AAVS1 safe harbor locus.

[0040] In one embodiment, the transgene is a chemotherapy selection marker, a cell surface antigen, or a suicide gene. In one embodiment, the chemotherapy selection marker is a gene encoding the P140K mutant form of methylguanine methyltransferase (P140K). In another embodiment, the cell surface antigen is a gene encoding truncated CD19 (tCD19) or a gene encoding truncated CD20 (tCD20). In another embodiment, the suicide gene is a gene encoding tCD20 or an inducible caspase-9 transgene (iCaspase-9).

[0041] In one embodiment, the transgene is a gene encoding P140K, a gene encoding tCD19, a gene encoding tCD20, or a gene encoding iCaspase-9.

[0042] In another embodiment, the cell is contacted with a plurality of transgenes. In one embodiment, the plurality of transgenes is integrated into the genome of the cell. In another embodiment, the plurality of transgenes is integrated into a safe harbor locus within the genome of the cell. In one embodiment, the safe harbor locus is the AAVS1 safe harbor locus.

[0043] In one embodiment, the plurality of transgenes comprises two, three, or all of the following: a gene encoding a P140K mutant form of methylguanine methyltransferase, a gene encoding tCD19, a gene encoding tCD20, or iCaspase-9. In another embodiment, the plurality of transgenes comprises or consists of a gene encoding a P140K mutant form of methylguanine methyltransferase and a gene encoding tCD20.

[0044] In one embodiment, the method further comprises contacting the cell with an enzymatically active (eiCas9) molecule. In one embodiment, the eiCas9 is fused to a transcriptional repressor or a transcriptional activator.

[0045] In one embodiment, the gRNA molecule comprises a targeting domain that is complementary to a target domain in a target gene. In one embodiment, the target gene is listed in Table 4.

[0046] In one aspect, disclosed herein are cells modified by the methods disclosed herein.

[0047] In another aspect, disclosed herein is a pharmaceutical composition comprising the cells disclosed herein.

[0048] In one aspect, disclosed herein is a method of treating or preventing a disease in a subject, comprising administering to the subject modified cells, or cells that have been altered by the methods disclosed herein.

[0049] In one aspect, a stem cell gene editing system is disclosed herein, comprising a stem cell viability enhancer; a gRNA molecule; and a Cas9 molecule. The gRNA molecule may be a modified gRNA molecule. In one embodiment, the stem cell gene editing system further comprises a stem cell, wherein the stem cell comprises a gRNA molecule and a Cas9 molecule. In one embodiment, the stem cell gene editing system further comprises a stem cell, wherein the stem cell comprises a stem cell viability enhancer.

[0050] In one embodiment, the stem cell gene editing system is a kit comprising each of the components. In another embodiment, the stem cell gene editing system is a composition. In one embodiment, the composition is part of a kit. In one embodiment, the kit further comprises instructions for modifying a target nucleic acid in a stem cell.

[0051] In one embodiment, the stem cell viability enhancer is selected from the group consisting of an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist. In one embodiment, the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), AhRA, dimethoxyflavone, 6,2',4'-trimethoxyflavone, LGC0006, α-naphthoflavone, and CH-223191. In one embodiment, the AhR antagonist is SR1. In one embodiment, the innate immune response antagonist is selected from the group consisting of cyclosporin A, dexamethasone, resveratrol, MyD88 inhibitory peptide, RNAi agent targeting Myd88, B18R recombinant protein, glucocorticoid, OxPAPC, TLR antagonist, rapamycin, BX795, and RLR inhibitor. In one embodiment, the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16,16-dimethylprostaglandin E2 (dmPGE2).

[0052] In one embodiment, the modified gRNA molecule comprises a 5'-terminal cap structure. In one embodiment, the modified gRNA molecule comprises a 3'-terminal polyadenine tail. In one embodiment, the modified gRNA molecule comprises a 5'-terminal cap structure and a 3'-terminal polyadenine tail.

[0053] In one embodiment, the Cas9 molecule is selected from the group consisting of wild-type Cas9, nickase Cas9, inactive Cas9 (dCas9), split Cas9, and inducible Cas9. In one embodiment, the Cas9 molecule is enzymatically active Cas9 (eaCas9).

[0054] In one embodiment, the Cas9 molecule comprises N-terminal RuvC-like domain cleavage activity but not HNH-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position N863 of Streptococcus pyogenes Cas9. In one embodiment, the Cas9 molecule comprises HNH-like domain cleavage activity but not N-terminal RuvC-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

[0055] In one embodiment, the Cas9 molecule is a Cas9 polypeptide. In one embodiment, the modified gRNA molecule and the Cas9 polypeptide are combined in a preformed ribonucleotide complex. In one embodiment, the Cas9 molecule is a nucleic acid encoding the Cas9 polypeptide.

[0056] In one embodiment, the stem cell editing system further comprises a cytokine, hi one embodiment, the cytokine is selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

[0057] In one embodiment, the composition further comprises one or more of the following: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-β signaling modulator, insulin-like growth factor binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), angiopoietin (ANG1), angiopoietin-like protein (ANGPTL4), an SDF1 / CXCR4 transduction axis modulator, or a Wnt signaling modulator.

[0058] In one embodiment, the stem cell editing system further comprises a template nucleic acid. In one embodiment, the template nucleic acid is a single-stranded oligodeoxynucleotide (ssODN). In one embodiment, the ssODN comprises a 5' phosphorothioate modification, a 3' phosphorothioate modification, or both a 5' phosphorothioate modification and a 3' phosphorothioate modification.

[0059] In one embodiment, the gRNA molecule comprises a targeting domain that is complementary to a target domain in a target gene. In one embodiment, the target gene is listed in Table 4.

[0060] In one aspect, disclosed herein are cells comprising the compositions disclosed herein.

[0061] In another aspect, disclosed herein is a pharmaceutical composition comprising a composition disclosed herein and a pharmaceutically acceptable carrier. In another aspect, disclosed herein is a pharmaceutical composition comprising a cell disclosed herein and a pharmaceutically acceptable carrier.

[0062] In one embodiment, the HSC cells differentiate in vivo after transplantation. In one embodiment, the HSC cells differentiate into B cells, T cells, erythroid cells, and / or myeloid cells. In one embodiment, the HSC cells reconstitute hematopoiesis in the subject.

[0063] In one embodiment, the cells are implanted into the subject by intravenous infusion.

[0064] In one embodiment, one or more contacting steps are performed in vitro.

[0065] In one embodiment, the method further comprises generating the cells from iPS cells or from endothelial cells.

[0066] In one embodiment, the one or more cell viability enhancers have one or more of the following properties: enhance cell maintenance, increase cell lifespan, enhance cell viability, or increase cell proliferation. In another embodiment, the one or more cell viability enhancers have one or more of the following properties: inhibit differentiation, inhibit apoptotic cell death, inhibit necrosis, inhibit autophagy, or inhibit senescence. In one embodiment, the one or more cell viability enhancers inhibit senescence associated with the DNA damage response.

[0067] In one embodiment, the one or more cell viability enhancers inhibit the innate immune response and / or prevent apoptosis of cells. In another embodiment, the one or more cell viability enhancers inhibit the innate immune response and / or prevent apoptosis of cells in response to CRISPR / Cas9 components. In another embodiment, the one or more cell viability enhancers inhibit an interferon or toll-like receptor response to an exogenous nucleic acid.

[0068] In one embodiment, the method further comprises contacting the cells with a chemotherapeutic agent to expand cell number. In one embodiment, the cells comprise a chemotherapeutic selectable marker. In one embodiment, the chemotherapeutic selectable marker is the P140K mutant form of methylguanine methyltransferase. In one embodiment, the chemotherapeutic agent is O6BG / BCNU. In one embodiment, the contacting step is performed in vitro or in vivo.

[0069] In one embodiment, the method further comprises contacting the cells with a cell surface antigen binding agent for selection of the cells. In one embodiment, the cells comprise a cell surface antigen. In another embodiment, the cell surface antigen is tCD19 or tCD20. In one embodiment, the cell surface antigen is a tCD19-binding reagent or a tCD20-binding reagent. In one embodiment, the contacting step is performed in vitro or in vivo.

[0070] In one embodiment, the method further comprises contacting the cell with an agent that results in cell death, hi one embodiment, the cell is a HSC that contains a suicide gene.

[0071] In one embodiment, the method further comprises contacting the cells with an anti-CD20 antibody, hi one embodiment, the anti-CD20 antibody is rituximab.

[0072] In one embodiment, the method further comprises contacting the cells with a substance that dimerizes iCaspase-9, wherein the cells contain iCaspase-9. In one embodiment, the substance that dimerizes iCaspase-9 is a cell-permeable dimerizing agent. In another embodiment, the cell-permeable dimerizing agent is AP20187 or AP1903.

[0073] In one embodiment, the method further comprises contacting the cell with a template nucleic acid comprising a transgene and one or more gRNAs comprising a targeting domain complementary to a targeting domain from the region into which the transgene has integrated.

[0074] In one embodiment, the method further comprises culturing the cells under conditions that allow expression of the transgene.

[0075] In another embodiment, the eiCas9 molecule is a fusion molecule that regulates a target gene. In one embodiment, the target gene is a target gene that temporarily prevents cell death, increases cell lifespan, enhances cell viability, or increases proliferation. In another embodiment, the eiCas9 molecule is fused with a KRAB domain.

[0076] In one embodiment, expression of the transgene does not significantly reduce multipotency, cytocompatibility, or both. In another embodiment, expression of the transgene does not significantly reduce viability, multipotency, cytocompatibility, or both after brief exposure to CRISPR / Cas9 components.

[0077] In one embodiment, the cells are cultured under hypoxic culture conditions, which in one embodiment include 10% or less, 8% or less, 5% or less, 3% or less, 1% or less, or 0.5% or less O2.

[0078] In one embodiment, the cells are cultured in a three-dimensional culture system. In one embodiment, the three-dimensional culture system is a NANEX™ 3D culture system.

[0079] In one embodiment, the method further comprises co-culturing the cells with endothelial cells, mesenchymal cells, or both. In one embodiment, the endothelial cells are VeraVecs cells. In another embodiment, the mesenchymal cells are mesenchymal stromal cells or perivascular mesenchymal cells.

[0080] In one embodiment, the method further comprises washing the cells after the contacting step. In one embodiment, the method further comprises washing the cells between the contacting step with the cell viability enhancer and the contacting step with the gRNA molecule and the Cas9 molecule.

[0081] In one embodiment, the subject is the same subject from which the cells are isolated. In another embodiment, the subject is a different subject from which the cells are isolated.

[0082] In another aspect, disclosed herein are methods for treating or preventing a disease in a subject, comprising administering to the subject modified cells or cells altered by the methods disclosed herein. In one embodiment, the subject is suffering from or at risk of developing a disease listed in Table 4. In another embodiment, the disease is a hemoglobinopathy, anemia, a hemostatic disorder, a metabolic disorder, a severe immunodeficiency, a myeloid immunodeficiency, a B lymphocyte and immunoglobulin immunodeficiency, a cytopenic disorder, a metabolic enzyme deficiency, a transport and storage disease, an erythroid disorder, an autoimmune disease, an inflammatory disease, an infectious disease, or a neoplastic disease. In one embodiment, the cytopenic disorder has neurological complications. In another embodiment, the neoplastic disease is lymphoma or leukemia.

[0083] In one embodiment, about 1 x 10 per kg of body weight 5 ~Approx. 1×10 8 In another embodiment, about 1 x 10 altered or modified cells per kg of body weight are administered to the subject. 6 ~Approx. 1×10 7 In another embodiment, about 1 x 10 altered or modified cells per kg of body weight are administered to the subject. 6 , about 2×10 6 , or about 5 × 10 6 The altered or modified cells are administered to the subject.

[0084] In one embodiment, the cells are used in the manufacture of a medicament for treating or preventing a disease. In one embodiment, the disease is a disease listed in Table 4.

[0085] In one embodiment, the cells are hematopoietic stem / progenitor cells (HSCs). In one embodiment, the HSCs are capable of differentiation in vivo after transplantation into a subject. In one embodiment, the HSCs are capable of differentiating into B cells, T cells, erythroid cells, and / or myeloid cells. In another embodiment, the HSCs are capable of reconstituting hematopoiesis in a subject.

[0086] In another embodiment, disclosed herein is a reaction mixture that includes one, two, or all of the following: (a) cells; (b) one or more CRISPR / Cas9 components; and (c) one, two, or all of: (i) a cell viability enhancer; (ii) a transgene; or (iii) an eiCas9 molecule.

[0087] In one embodiment, the one or more CRISPR / Cas9 components comprise a Cas9 molecule, a gRNA molecule, or both. In another embodiment, the reaction mixture further comprises a donor template nucleic acid. In another embodiment, the eiCas9 molecule is fused to a transcriptional repressor or a transcriptional activator.

[0088] In another aspect, disclosed herein is a kit comprising: (a) one, two, or all of: (i) a cell viability enhancer; (ii) a transgene; or (iii) an eiCas9 molecule; and (b) instructions for modifying a cell or generating a modified cell.

[0089] In one embodiment, the eiCas9 molecule is fused to a transcriptional repressor or a transcriptional activator. In another embodiment, the cell is a HSC.

[0090] In another aspect, disclosed herein is the use of a cell in the manufacture of a medicament for treating or preventing a disease. In one embodiment, the disease is a disease listed in Table 4.

[0091] In one aspect, disclosed herein is the use of a cell described herein in the manufacture of a medicament for treating or preventing a disease, e.g., a disease described herein, e.g., a disease listed in Table 4.

[0092] In one aspect, disclosed herein is a cell as described herein for treating or preventing a disease, e.g., a disease described herein, e.g., a disease listed in Table 4.

[0093] As disclosed herein, the compositions, reaction mixtures, and kits can also include a control gRNA molecule, such as, for example, a control gRNA molecule disclosed herein.

[0094] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials similar or equivalent to those described herein can be used in the present invention or in the testing of the embodiments of this disclosure, and suitable methods and materials are listed below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In addition, materials, methods and examples are intended to be illustrative only and are not intended to be limiting.

[0095] Headings, including numerical and alphabetical headings and subheadings, are for organizational and presentation purposes only and are not intended to be limiting.

[0096] Other features and advantages of the disclosed embodiments will become apparent from the detailed description, the drawings, and the claims. [Brief explanation of the drawings]

[0097] [Figure 1] Depicts the detection of indels in the CCR5 locus after delivery of S. aureus gRNA and S. aureus Cas9. [Figure 2] Depicts flow cytometry analysis of genome-edited HSCs to determine co-expression of stem cell phenotypic markers CD34 and CD90 and viability (7-AAD-Annexin V-cells). [Figure 3A] Depicts fold change in number (e.g., maintenance of viability) of Nucleofected™ CD34+ cells 96 hours after delivery of the indicated Cas9 variants in combination with CXCR4 gRNA or GFP-expressing plasmid alone (pmaxGFP). Treatment + / - 40 nM UM171 is indicated by a minus sign (-, no UM171) or a plus sign (+, with 40 nM UM171). [Figure 3B] Figure 1 depicts the percentage of indels detected by T7E1 assay in CD34+ HSCs after the indicated Nucleofection™ treatments ±40 nM UM171 are indicated by a minus sign (-, no UM171) or a plus sign (+, with 40 nM UM171). [Figure 4A] 1 depicts the fold change in the number (e.g., maintenance of viability and proliferative capacity) of Nucleofected™ CD34+ cells 96 hours after co-delivery of Cas9 in combination with CXCR4 gRNA (CXCR4-231) and CCR5 gRNA (CCR5-U43) plasmids. [Figure 4B]1 depicts the percentage of indels detected by the T7E1 assay in CD34+ HSCs at the CCR5 and CXCR4 genomic loci. [Figure 5A] Depicting the kinetics of fold change in CD34+ cell numbers after electroporation with the indicated uncapped / tailed gRNAs or capped / tailed gRNAs with combined Cas9 mRNA (either S. pyogenes (Sp) or S. aureus (S. aureus) Sa Cas9). [Figure 5B] Depicts the fold change in total viable CD34+ cells 72 hours after electroporation with the indicated uncapped / tailed gRNA or capped / tailed gRNA with combined Cas9 mRNA (either S. pyogenes (Sp) or S. aureus (S. aureus) Sa Cas9). [Figure 5C] Depicts representative flow cytometry data showing the maintenance of viable (propidium iodide negative) human CD34+ cells after electroporation with capped and tailed AAVS1 gRNA and Cas9 mRNA. [Figure 6A] Depicts the percentage of insertions / deletions (indels) detected in CD34+ cells and their hematopoietic colony-forming cell (CFC) progeny at the targeted AAVS1 locus following delivery of capped and tailed AAVS1 gRNA and Cas9 mRNA compared to uncapped and untailed AAVS1 gRNA. [Figure 6B] Depicts maintenance of hematopoietic colony-forming potential (CFC) in CD34+ cells after editing with capped / tailed AAVS1 gRNA. Note the loss of CFC capacity in cells electroporated with uncapped / tailed AAVS1 gRNA. E: erythrocyte, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte. [Figure 6C]We describe efficient targeted locus editing (% indels) in the K562 erythroleukemia cell line, a human erythroleukemia cell line with properties similar to HSCs after delivery of capped and tailed HBB gRNA together with S. pyogenes Cas9 mRNA or ribonucleoprotein (RNP). [Figure 6D] Depicts Cas9-mediated / capped-and-tailed gRNA-mediated editing (% indels) at the indicated targeted loci (AAVS1, HBB, CXCR4) in human cord blood CD34+ cells. Right: CFC capacity of cord blood CD34+ cells after electroporation with Cas9 mRNA and capped-and-tailed HBB-8 gRNA (also referred to herein as HBB_Sp8) (SEQ ID NO: 388) (non-electroporated control or cells electroporated with 2 or 10 μg HBB gRNA). Cells were electroporated with Cas9 mRNA or 2 or 10 μg gRNA. [Figure 6E] Depicts CFC assay of cells electroporated with 2 μg or 10 μg of capped / tailed HBB gRNA. CFC: colony forming cells, E: erythrocytes, G: granulocytes, M: macrophages, GM: granulocyte-macrophages, GEMM: granulocyte-erythrocyte-macrophage-monocytes. [Figure 6F] Figure 6 depicts a representative gel image (T7E1 analysis) showing cleavage at the indicated loci in cord blood CD34+ cells 72 hours after delivery of capped and tailed AAVS1, HBB, or CXCR4 gRNA and S. pyogenes Cas9 mRNA. This exemplary gel corresponds to the summary data shown in Figure 6D. [Figure 6G] 1 shows cell viability in CB CD34+ cells 48 hours after delivery of Cas9 mRNA and the indicated gRNAs, as determined by co-staining with 7-AAD and Annexin V and flow cytometry analysis. [Figure 7A]Figure 7 depicts analysis of the stability of D10A nickase RNP in human adult CD34+ HSCs in vitro and ex vivo. Figure 7A depicts a differential scanning fluorometric shift assay after complexing D10A protein with the indicated HBB gRNAs added at a 1:1 molar ratio of gRNA:RNP. Figure 7B depicts detection of Cas9 protein in cell lysates 72 hours after electroporation of human adult CD34+ HSCs with D10A nickase RNP or with D10A mRNA containing gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387). The electroporation program used (P2 or P3) is indicated at the top of the image. 2x gRNA: 10 μg of each gRNA was co-delivered with D10A mRNA (5 μg of each gRNA). [Figure 7B] Figure 7 depicts analysis of the stability of D10A nickase RNP in human adult CD34+ HSCs in vitro and ex vivo. Figure 7A depicts a differential scanning fluorometric shift assay after complexing D10A protein with the indicated HBB gRNAs added at a 1:1 molar ratio of gRNA:RNP. Figure 7B depicts detection of Cas9 protein in cell lysates 72 hours after electroporation of human adult CD34+ HSCs with D10A nickase RNP or with D10A mRNA containing gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387). The electroporation program used (P2 or P3) is indicated at the top of the image. 2x gRNA: 10 μg of each gRNA was co-delivered with D10A mRNA (5 μg of each gRNA). [Figure 8A]Figure 8A shows that human adult CD34+ HSCs maintain stem cell phenotype after electroporation with the D10A nickase RNP and HBB-targeting gRNA pair. Figure 8A shows that gene-edited adult CD34+ cells maintain expression of stem cell markers CD34 and CD133 72 hours after electroporation. Figure 8B depicts absolute viable (7-AAD-annexin V-) CD34+ cell counts at the indicated time points following electroporation of the D10A RNP HBB gRNA pair. Figure 8C shows that gene-edited adult CD34+ cells maintain hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. [Figure 8B] Figure 8A shows that human adult CD34+ HSCs maintain stem cell phenotype after electroporation with the D10A nickase RNP and HBB-targeting gRNA pair. Figure 8A shows that gene-edited adult CD34+ cells maintain expression of stem cell markers CD34 and CD133 72 hours after electroporation. Figure 8B depicts absolute viable (7-AAD-annexin V-) CD34+ cell counts at the indicated time points following electroporation of the D10A RNP HBB gRNA pair. Figure 8C shows that gene-edited adult CD34+ cells maintain hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. [Figure 8C]Figure 8A shows that human adult CD34+ HSCs maintain stem cell phenotype after electroporation with the D10A nickase RNP and HBB-targeting gRNA pair. Figure 8A shows that gene-edited adult CD34+ cells maintain expression of stem cell markers CD34 and CD133 72 hours after electroporation. Figure 8B depicts absolute viable (7-AAD-annexin V-) CD34+ cell counts at the indicated time points following electroporation of the D10A RNP HBB gRNA pair. Figure 8C shows that gene-edited adult CD34+ cells maintain hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. [Figure 9A] Figure 9A shows that D10A nickase RNP co-delivered with an HBB-targeting gRNA pair supports gene editing and HDR in human CD34+ HSCs. Figure 9A depicts the percentage of gene editing events detected by T7E1 nuclease assay analysis of the HBB locus in human adult CD34+ HSCs. Figure 9B depicts DNA sequencing analysis of the HBB locus from human adult CD34+ HSCs. Gene editing event subtypes (insertion, deletion, indel, and gene conversion events) are displayed. RNP* refers to the use of an alternate electroporation program (P3). Figure 9C depicts the percentage of editing event types detected in gDNA from human adult CD34+ HSCs electroporated under the conditions shown in Figure 9B. Data are presented as a percentage of all gene editing events. [Figure 9B]Figure 9A shows that D10A nickase RNP co-delivered with an HBB-targeting gRNA pair supports gene editing and HDR in human CD34+ HSCs. Figure 9A depicts the percentage of gene editing events detected by T7E1 nuclease assay analysis of the HBB locus in human adult CD34+ HSCs. Figure 9B depicts DNA sequencing analysis of the HBB locus from human adult CD34+ HSCs. Gene editing event subtypes (insertion, deletion, indel, and gene conversion events) are displayed. RNP* refers to the use of an alternate electroporation program (P3). Figure 9C depicts the percentage of editing event types detected in gDNA from human adult CD34+ HSCs electroporated under the conditions shown in Figure 9B. Data are presented as a percentage of all gene editing events. [Figure 9C] Figure 9A shows that D10A nickase RNP co-delivered with an HBB-targeting gRNA pair supports gene editing and HDR in human CD34+ HSCs. Figure 9A depicts the percentage of gene editing events detected by T7E1 nuclease assay analysis of the HBB locus in human adult CD34+ HSCs. Figure 9B depicts DNA sequencing analysis of the HBB locus from human adult CD34+ HSCs. Gene editing event subtypes (insertion, deletion, indel, and gene conversion events) are displayed. RNP* refers to the use of an alternate electroporation program (P3). Figure 9C depicts the percentage of editing event types detected in gDNA from human adult CD34+ HSCs electroporated under the conditions shown in Figure 9B. Data are presented as a percentage of all gene editing events. [Figure 10]Depicts flow cytometry analysis of β-hemoglobin expression in erythroid progeny differentiated from D10A nickase RNP gene-edited adult CD34+ HSCs. CFU-E colonies (far left) differentiated from CD34+ cells electroporated with D10A RNP HBB gRNA were dissociated, fixed, permeabilized, and stained for β-hemoglobin expression. The frequency of gene editing detected in the parental CD34+ cell population is displayed above the histogram for the indicated sample. The percentage of β-hemoglobin expression in each colony was determined by flow cytometry and is displayed above the right side of each histogram. [Figure 11A] Figure 11A shows that human umbilical cord blood (CB) CD34+ HSCs maintained stem cell phenotype after electroporation with the D10A nickase RNP and HBB-targeting gRNA pair. Figure 11A shows that gene-edited human CB CD34+ HSC cells maintained viability after electroporation. Right: Absolute viable (7-AAD-annexin V-) human CB CD34+ HSC cell counts at the indicated time points for electroporation with the D10A RNP HBB gRNA pair. Figure 11B depicts that gene-edited CB CD34+ cells maintained hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. The amount of D10A RNP delivered per million cells (5 or 10 μg) and the recovery temperature 2 hours after electroporation of parental CB CD34+ cells (in brackets) are indicated. [Figure 11B]Figure 11A shows that human umbilical cord blood (CB) CD34+ HSCs maintained stem cell phenotype after electroporation with the D10A nickase RNP and HBB-targeting gRNA pair. Figure 11A shows that gene-edited human CB CD34+ HSC cells maintained viability after electroporation. Right: Absolute viable (7-AAD-annexin V-) human CB CD34+ HSC cell counts at the indicated time points for electroporation with the D10A RNP HBB gRNA pair. Figure 11B depicts that gene-edited CB CD34+ cells maintained hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. The amount of D10A RNP delivered per million cells (5 or 10 μg) and the recovery temperature 2 hours after electroporation of parental CB CD34+ cells (in brackets) are indicated. [Figure 12A] Figure 12A shows that D10A nickase RNP co-delivered with an HBB-targeting gRNA pair supported gene editing and HDR in human CB CD34+ HSCs. Figure 12A depicts the percentage of gene editing events detected by T7E1 endonuclease assay analysis of the HBB locus in gene-edited human CB CD34+ HSCs. Figure 12B depicts DNA sequence analysis of the HBB locus in gene-edited human CB CD34+ HSCs. Gene editing event subtypes (insertion, deletion, indel, and gene conversion events) are displayed as a fraction of total sequencing reads. Figure 12C depicts the gene editing event subtypes expressed as a relative percentage of the total gene editing events detected. The amount of D10A RNP delivered per million cells (5 or 10 μg) and the 2-hour recovery temperature (in parentheses) after electroporation of parental CB CD34+ HSCs are displayed. [Figure 12B]Figure 12A shows that D10A nickase RNP co-delivered with an HBB-targeting gRNA pair supported gene editing and HDR in human CB CD34+ HSCs. Figure 12A depicts the percentage of gene editing events detected by T7E1 endonuclease assay analysis of the HBB locus in gene-edited human CB CD34+ HSCs. Figure 12B depicts DNA sequence analysis of the HBB locus in gene-edited human CB CD34+ HSCs. Gene editing event subtypes (insertion, deletion, indel, and gene conversion events) are displayed as a fraction of total sequencing reads. Figure 12C depicts the gene editing event subtypes expressed as a relative percentage of the total gene editing events detected. The amount of D10A RNP delivered per million cells (5 or 10 μg) and the 2-hour recovery temperature (in parentheses) after electroporation of parental CB CD34+ HSCs are displayed. [Figure 12C] Figure 12A shows that D10A nickase RNP co-delivered with an HBB-targeting gRNA pair supported gene editing and HDR in human CB CD34+ HSCs. Figure 12A depicts the percentage of gene editing events detected by T7E1 endonuclease assay analysis of the HBB locus in gene-edited human CB CD34+ HSCs. Figure 12B depicts DNA sequence analysis of the HBB locus in gene-edited human CB CD34+ HSCs. Gene editing event subtypes (insertion, deletion, indel, and gene conversion events) are displayed as a fraction of total sequencing reads. Figure 12C depicts the gene editing event subtypes expressed as a relative percentage of the total gene editing events detected. The amount of D10A RNP delivered per million cells (5 or 10 μg) and the 2-hour recovery temperature (in parentheses) after electroporation of parental CB CD34+ HSCs are displayed. [Figure 13A]Figure 13 depicts directed differentiation of gene-edited human CB CD34+ HSCs into erythroblasts. Flow cytometry analysis of day 18 erythroblasts differentiated from gene-edited human CB CD34+ HSCs. Figure 13A depicts CD71 (transferrin receptor) and CD235 (glycophorin A). Figure 13B depicts fetal hemoglobin (g hemoglobin). Figure 13C depicts loss of CD45 and dsDNA due to enucleation, as indicated by the absence of dsDNA (negative for the dsDNA-binding dye DRAQ5). Unlike adult CD34+ cells, CB CD34+ cells differentiate into fetal-like erythroblasts that express fetal g hemoglobin (but not adult b hemoglobin). [Figure 13B] Figure 13 depicts directed differentiation of gene-edited human CB CD34+ HSCs into erythroblasts. Flow cytometry analysis of day 18 erythroblasts differentiated from gene-edited human CB CD34+ HSCs. Figure 13A depicts CD71 (transferrin receptor) and CD235 (glycophorin A). Figure 13B depicts fetal hemoglobin (g hemoglobin). Figure 13C depicts loss of CD45 and dsDNA due to enucleation, as indicated by the absence of dsDNA (negative for the dsDNA-binding dye DRAQ5). Unlike adult CD34+ cells, CB CD34+ cells differentiate into fetal-like erythroblasts that express fetal g hemoglobin (but not adult b hemoglobin). [Figure 13C] Figure 13 depicts directed differentiation of gene-edited human CB CD34+ HSCs into erythroblasts. Flow cytometry analysis of day 18 erythroblasts differentiated from gene-edited human CB CD34+ HSCs. Figure 13A depicts CD71 (transferrin receptor) and CD235 (glycophorin A). Figure 13B depicts fetal hemoglobin (g hemoglobin). Figure 13C depicts loss of CD45 and dsDNA due to enucleation, as indicated by the absence of dsDNA (negative for the dsDNA-binding dye DRAQ5). Unlike adult CD34+ cells, CB CD34+ cells differentiate into fetal-like erythroblasts that express fetal g hemoglobin (but not adult b hemoglobin). [Figure 14A]Figure 14A depicts that human CB CD34+ HSCs maintain stem cell phenotype after electroporation with Cas9 mutant RNP and HBB-targeting gRNA pairs. Figure 14A depicts that gene-edited human CB CD34+ cells maintained viability after electroporation with WTCas9 endotoxin-free (EF WT) Cas9, N863A nickase, or D10A nickase co-delivered with the HBB gRNA pair. Absolute viable (7-AAD-annexin V-) CD34+ HSC cell counts at the indicated time points relative to electroporation. Figure 14B depicts that gene-edited CB CD34+ cells maintained hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. The amount of RNP delivered per million cells (10 μg) and the recovery temperature 2 hours after electroporation of parental human CB CD34+ cells (in brackets) are indicated. [Figure 14B] Figure 14A depicts that human CB CD34+ HSCs maintain stem cell phenotype after electroporation with Cas9 mutant RNP and HBB-targeting gRNA pairs. Figure 14A depicts that gene-edited human CB CD34+ cells maintained viability after electroporation with WTCas9 endotoxin-free (EF WT) Cas9, N863A nickase, or D10A nickase co-delivered with the HBB gRNA pair. Absolute viable (7-AAD-annexin V-) CD34+ HSC cell counts at the indicated time points relative to electroporation. Figure 14B depicts that gene-edited CB CD34+ cells maintained hematopoietic colony-forming cell (CFC) activity and multipotency. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte CFC. The amount of RNP delivered per million cells (10 μg) and the recovery temperature 2 hours after electroporation of parental human CB CD34+ cells (in brackets) are indicated. [Figure 15A]Figure 15A depicts a comparison of gene editing at the HBB locus in human CB CD34+ cells mediated by WT and nickase Cas9 mutant RNPs. Figure 15A depicts T7E1 analysis of the percentage of indels detected 72 hours after electroporation at targeted sites in the HBB locus after electroporation of WT Cas9, endotoxin-free WT Cas9 (EF-WT), N863A nickase, and D10A nickase RNPs (each co-delivered with the HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNA pairs). Figure 15B depicts Western blot analysis showing detection of Cas9 mutants in cell lysates of CB CD34+ cells at the indicated time points after electroporation. The amount of RNP delivered per million cells (10 μg) and the 2-hour recovery temperature after electroporation of parental CB CD34+ cells (in parentheses) are shown. [Figure 15B] Figure 15A depicts a comparison of gene editing at the HBB locus in human CB CD34+ cells mediated by WT and nickase Cas9 mutant RNPs. Figure 15A depicts T7E1 analysis of the percentage of indels detected 72 hours after electroporation at targeted sites in the HBB locus after electroporation of WT Cas9, endotoxin-free WT Cas9 (EF-WT), N863A nickase, and D10A nickase RNPs (each co-delivered with the HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNA pairs). Figure 15B depicts Western blot analysis showing detection of Cas9 mutants in cell lysates of CB CD34+ cells at the indicated time points after electroporation. The amount of RNP delivered per million cells (10 μg) and the 2-hour recovery temperature after electroporation of parental CB CD34+ cells (in parentheses) are shown. [Figure 16A]Figure 16A depicts a comparison of HDR and NHEJ events detected at the HBB locus after gene editing using WTCas9 and D10A nickase in human CB CD34+ HSCs. Figure 16A depicts the percentage of gene editing events (72 hours after electroporation) detected by DNA sequencing analysis and shown as a percentage of total sequence reads. CB CD34+ HSC cells received RNPs (WTCas9, endotoxin-free WTCas9 (EF-WT), N863A nickase, and D10A nickase) along with the HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNA pair. Figure 16B depicts the percentage of each type of editing event detected in gDNA from cells electroporated under the conditions shown in Figure 16A. Data are shown as a percentage of total gene editing events. From left to right: 10 μg of WTTas9RNP (37°C), 10 μg of endotoxin-free (EF) WTTas9RNP (37°C), 10 μg of D10A Cas9RNP (37°C), 10 μg of D10A Cas9RNP (30°C). [Figure 16B] Figure 16A depicts a comparison of HDR and NHEJ events detected at the HBB locus after gene editing using WTCas9 and D10A nickase in human CB CD34+ HSCs. Figure 16A depicts the percentage of gene editing events (72 hours after electroporation) detected by DNA sequencing analysis and shown as a percentage of total sequence reads. CB CD34+ HSC cells received RNPs (WTCas9, endotoxin-free WTCas9 (EF-WT), N863A nickase, and D10A nickase) along with the HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNA pair. Figure 16B depicts the percentage of each type of editing event detected in gDNA from cells electroporated under the conditions shown in Figure 16A. Data are shown as a percentage of total gene editing events. From left to right: 10 μg of WTTas9RNP (37°C), 10 μg of endotoxin-free (EF) WTTas9RNP (37°C), 10 μg of D10A Cas9RNP (37°C), 10 μg of D10A Cas9RNP (30°C). [Figure 17A]Figure 17 depicts in vitro transcribed HBB-specific gRNAs generated with polyA tails encoded in the DNA templates. Figure 17A depicts PCR products of DNA templates for HBB gRNAs with encoded polyA tails of the indicated lengths. Predominant size-appropriate PCR products for gRNAs with 10-, 20-, and 50-length polyA tails are shown in solid boxes. The distribution of PCR products is shown in dotted boxes. Figure 17B depicts bioanalyzer analysis of in vitro transcribed gRNAs with the indicated tail lengths either modified in the DNA templates or enzymatically added (E-PAP). [Figure 17B] Figure 17 depicts in vitro transcribed HBB-specific gRNAs generated with polyA tails encoded in the DNA templates. Figure 17A depicts PCR products of DNA templates for HBB gRNAs with encoded polyA tails of the indicated lengths. Predominant size-appropriate PCR products for gRNAs with 10-, 20-, and 50-length polyA tails are shown in solid boxes. The distribution of PCR products is shown in dotted boxes. Figure 17B depicts bioanalyzer analysis of in vitro transcribed gRNAs with the indicated tail lengths either modified in the DNA templates or enzymatically added (E-PAP). [Figure 18A]Figure 18A depicts modified gRNAs with 10 and 20 polyA tails supported gene editing in human CB CD34+ HSCs. The left panel of Figure 18A depicts representative flow cytometry analysis plots showing viability (Annexin V-7AAD-) in human CB CD34+ HSCs 72 hours after electroporation with D10A Cas9 RNPs containing HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs. Right panel: Kinetics of fold change in CD34+ cell numbers after electroporation with D10A RNPs with the indicated polyA tails and HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs. Figure 18B depicts the percent viability (Annexin V-7AAD-) of cord blood CD34+ HSCs 72 hours after electroporation with D10ARNP and modified HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs with the indicated tail lengths. Figure 18C depicts gene editing detected by T7E1 endonuclease assay and Sanger DNA sequencing of PCR products of the HBB genomic locus in human CB CD34+ HSCs after electroporation with D10ARNP and modified HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs with the indicated polyA tails. [Figure 18B]Figure 18A depicts modified gRNAs with 10 and 20 polyA tails supported gene editing in human CB CD34+ HSCs. The left panel of Figure 18A depicts representative flow cytometry analysis plots showing viability (Annexin V-7AAD-) in human CB CD34+ HSCs 72 hours after electroporation with D10A Cas9 RNPs containing HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs. Right panel: Kinetics of fold change in CD34+ cell numbers after electroporation with D10A RNPs with the indicated polyA tails and HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs. Figure 18B depicts the percent viability (Annexin V-7AAD-) of cord blood CD34+ HSCs 72 hours after electroporation with D10ARNP and modified HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs with the indicated tail lengths. Figure 18C depicts gene editing detected by T7E1 endonuclease assay and Sanger DNA sequencing of PCR products of the HBB genomic locus in human CB CD34+ HSCs after electroporation with D10ARNP and modified HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs with the indicated polyA tails. [Figure 18C]Figure 18A depicts modified gRNAs with 10 and 20 polyA tails supported gene editing in human CB CD34+ HSCs. The left panel of Figure 18A depicts representative flow cytometry analysis plots showing viability (Annexin V-7AAD-) in human CB CD34+ HSCs 72 hours after electroporation with D10A Cas9 RNPs containing HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs. Right panel: Kinetics of fold change in CD34+ cell numbers after electroporation with D10A RNPs with the indicated polyA tails and HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs. Figure 18B depicts the percent viability (Annexin V-7AAD-) of cord blood CD34+ HSCs 72 hours after electroporation with D10ARNP and modified HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs with the indicated tail lengths. Figure 18C depicts gene editing detected by T7E1 endonuclease assay and Sanger DNA sequencing of PCR products of the HBB genomic locus in human CB CD34+ HSCs after electroporation with D10ARNP and modified HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) gRNAs with the indicated polyA tails. [Figure 19A]We demonstrate that CRISPR / Cas9 RNP supports highly efficient gene editing at the HBB locus in human adult and cord blood CD34+ hematopoietic stem / progenitor cells from 15 different stem cell donors. (Figure 19A) Summary of gene editing results determined by DNA sequencing of n=15 CD34+ cell donors and composite data from 15 experiments. Gene editing is shown for cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cell donors. Cas9 variants (D10A nickase or wild-type, WT) are indicated. (Figure 19B) Summary of the types of editing events detected in experiments in which CD34+ cells (n=10 donors) were contacted with D10A nickase RNP and gRNA pairs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)). (Figure 19C) Fold change in the number of RNP-treated and paired untreated control CD34+ cells 2-3 days after electroporation. (Figure 19D) Composite summary of CFC data showing total colonies, erythroid, and myeloid subtypes differentiated from human CD34+ cells (n = 10 donors). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 19E) DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from (Figure 19F) adult mPB CD34+ cells and (Figure 19F) CB CD34+ cells. Means and standard deviations are shown in all plots. Paired t-tests were performed for each donor pair shown in the data in panels Figures 19A-19D. [Figure 19B]We demonstrate that CRISPR / Cas9 RNP supports highly efficient gene editing at the HBB locus in human adult and cord blood CD34+ hematopoietic stem / progenitor cells from 15 different stem cell donors. (Figure 19A) Summary of gene editing results determined by DNA sequencing of n=15 CD34+ cell donors and composite data from 15 experiments. Gene editing is shown for cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cell donors. Cas9 variants (D10A nickase or wild-type, WT) are indicated. (Figure 19B) Summary of the types of editing events detected in experiments in which CD34+ cells (n=10 donors) were contacted with D10A nickase RNP and gRNA pairs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)). (Figure 19C) Fold change in the number of RNP-treated and paired untreated control CD34+ cells 2-3 days after electroporation. (Figure 19D) Composite summary of CFC data showing total colonies, erythroid, and myeloid subtypes differentiated from human CD34+ cells (n = 10 donors). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 19E) DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from (Figure 19F) adult mPB CD34+ cells and (Figure 19F) CB CD34+ cells. Means and standard deviations are shown in all plots. Paired t-tests were performed for each donor pair shown in the data in panels Figures 19A-19D. [Figure 19C]We demonstrate that CRISPR / Cas9 RNP supports highly efficient gene editing at the HBB locus in human adult and cord blood CD34+ hematopoietic stem / progenitor cells from 15 different stem cell donors. (Figure 19A) Summary of gene editing results determined by DNA sequencing of n=15 CD34+ cell donors and composite data from 15 experiments. Gene editing is shown for cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cell donors. Cas9 variants (D10A nickase or wild-type, WT) are indicated. (Figure 19B) Summary of the types of editing events detected in experiments in which CD34+ cells (n=10 donors) were contacted with D10A nickase RNP and gRNA pairs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)). (Figure 19C) Fold change in the number of RNP-treated and paired untreated control CD34+ cells 2-3 days after electroporation. (Figure 19D) Composite summary of CFC data showing total colonies, erythroid, and myeloid subtypes differentiated from human CD34+ cells (n = 10 donors). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 19E) DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from (Figure 19F) adult mPB CD34+ cells and (Figure 19F) CB CD34+ cells. Means and standard deviations are shown in all plots. Paired t-tests were performed for each donor pair shown in the data in panels Figures 19A-19D. [Figure 19D]We demonstrate that CRISPR / Cas9 RNP supports highly efficient gene editing at the HBB locus in human adult and cord blood CD34+ hematopoietic stem / progenitor cells from 15 different stem cell donors. (Figure 19A) Summary of gene editing results determined by DNA sequencing of n=15 CD34+ cell donors and composite data from 15 experiments. Gene editing is shown for cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cell donors. Cas9 variants (D10A nickase or wild-type, WT) are indicated. (Figure 19B) Summary of the types of editing events detected in experiments in which CD34+ cells (n=10 donors) were contacted with D10A nickase RNP and gRNA pairs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)). (Figure 19C) Fold change in the number of RNP-treated and paired untreated control CD34+ cells 2-3 days after electroporation. (Figure 19D) Composite summary of CFC data showing total colonies, erythroid, and myeloid subtypes differentiated from human CD34+ cells (n = 10 donors). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 19E) DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from (Figure 19F) adult mPB CD34+ cells and (Figure 19F) CB CD34+ cells. Means and standard deviations are shown in all plots. Paired t-tests were performed for each donor pair shown in the data in panels Figures 19A-19D. [Figure 19E]We demonstrate that CRISPR / Cas9 RNP supports highly efficient gene editing at the HBB locus in human adult and cord blood CD34+ hematopoietic stem / progenitor cells from 15 different stem cell donors. (Figure 19A) Summary of gene editing results determined by DNA sequencing of n=15 CD34+ cell donors and composite data from 15 experiments. Gene editing is shown for cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cell donors. Cas9 variants (D10A nickase or wild-type, WT) are indicated. (Figure 19B) Summary of the types of editing events detected in experiments in which CD34+ cells (n=10 donors) were contacted with D10A nickase RNP and gRNA pairs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)). (Figure 19C) Fold change in the number of RNP-treated and paired untreated control CD34+ cells 2-3 days after electroporation. (Figure 19D) Composite summary of CFC data showing total colonies, erythroid, and myeloid subtypes differentiated from human CD34+ cells (n = 10 donors). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 19E) DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from (Figure 19F) adult mPB CD34+ cells and (Figure 19F) CB CD34+ cells. Means and standard deviations are shown in all plots. Paired t-tests were performed for each donor pair shown in the data in panels Figures 19A-19D. [Figure 19F]We demonstrate that CRISPR / Cas9 RNP supports highly efficient gene editing at the HBB locus in human adult and cord blood CD34+ hematopoietic stem / progenitor cells from 15 different stem cell donors. (Figure 19A) Summary of gene editing results determined by DNA sequencing of n=15 CD34+ cell donors and composite data from 15 experiments. Gene editing is shown for cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cell donors. Cas9 variants (D10A nickase or wild-type, WT) are indicated. (Figure 19B) Summary of the types of editing events detected in experiments in which CD34+ cells (n=10 donors) were contacted with D10A nickase RNP and gRNA pairs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)). (Figure 19C) Fold change in the number of RNP-treated and paired untreated control CD34+ cells 2-3 days after electroporation. (Figure 19D) Composite summary of CFC data showing total colonies, erythroid, and myeloid subtypes differentiated from human CD34+ cells (n = 10 donors). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 19E) DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from (Figure 19F) adult mPB CD34+ cells and (Figure 19F) CB CD34+ cells. Means and standard deviations are shown in all plots. Paired t-tests were performed for each donor pair shown in the data in panels Figures 19A-19D. [Figure 20A]Figure 20A shows the engraftment of Cas9 RNP gene-edited human CB CD34+ cells in an immune-deficient mouse model. (Figure 20A) Experimental schematic of RNP delivery to HSCs and transplantation and long-term tracking in NSG mice. (Figure 20B) Summary of the subtypes of editing events detected in an experiment in which fresh CD34+ cells were contacted with D10A nickase RNP and a gRNA pair (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) for use in HSC transplantation. (Figure 20C) Analysis of CFC hematopoietic activity of gene-edited HSCs (left), and DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for the detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from CFCs (right). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 20D) Left panel: Detection of indels by T7E1 assay in gDNA extracted from blood samples taken at various time points after cell infusion from mice transplanted with RNP-treated human CD34+ cells. Right panel: Detection of human cells in mouse peripheral blood samples. Each line corresponds to the level of gene editing detected in four individual mice transplanted with RNP-treated human CD34+ cells. Representative flow cytometry analysis of peripheral blood samples taken from RNP-treated mice 12 weeks after human HSC transplantation. Human CD45+ cells were distinguished from mouse CD45+ cells in the blood. Human lymphoid and myeloid lineages were detected within the human CD45+ cell gate (Figure 20E). Kinetics of hematopoietic reconstitution of NSG mice containing human CD45+ cells in recipients of RNP-treated and control untreated CD34+ cells from the same human HSC donor. Lower panel: Percentage of lymphoid and myeloid cells and erythroid progenitors detected within the human CD45+ cell gate in transplanted mice. [Figure 20B]Figure 20A shows the engraftment of Cas9 RNP gene-edited human CB CD34+ cells in an immune-deficient mouse model. (Figure 20A) Experimental schematic of RNP delivery to HSCs and transplantation and long-term tracking in NSG mice. (Figure 20B) Summary of the subtypes of editing events detected in an experiment in which fresh CD34+ cells were contacted with D10A nickase RNP and a gRNA pair (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) for use in HSC transplantation. (Figure 20C) Analysis of CFC hematopoietic activity of gene-edited HSCs (left), and DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for the detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from CFCs (right). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 20D) Left panel: Detection of indels by T7E1 assay in gDNA extracted from blood samples taken at various time points after cell infusion from mice transplanted with RNP-treated human CD34+ cells. Right panel: Detection of human cells in mouse peripheral blood samples. Each line corresponds to the level of gene editing detected in four individual mice transplanted with RNP-treated human CD34+ cells. Representative flow cytometry analysis of peripheral blood samples taken from RNP-treated mice 12 weeks after human HSC transplantation. Human CD45+ cells were distinguished from mouse CD45+ cells in the blood. Human lymphoid and myeloid lineages were detected within the human CD45+ cell gate (Figure 20E). Kinetics of hematopoietic reconstitution of NSG mice containing human CD45+ cells in recipients of RNP-treated and control untreated CD34+ cells from the same human HSC donor. Lower panel: Percentage of lymphoid and myeloid cells and erythroid progenitors detected within the human CD45+ cell gate in transplanted mice. [Figure 20C]Figure 20A shows the engraftment of Cas9 RNP gene-edited human CB CD34+ cells in an immune-deficient mouse model. (Figure 20A) Experimental schematic of RNP delivery to HSCs and transplantation and long-term tracking in NSG mice. (Figure 20B) Summary of the subtypes of editing events detected in an experiment in which fresh CD34+ cells were contacted with D10A nickase RNP and a gRNA pair (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) for use in HSC transplantation. (Figure 20C) Analysis of CFC hematopoietic activity of gene-edited HSCs (left), and DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for the detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from CFCs (right). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 20D) Left panel: Detection of indels by T7E1 assay in gDNA extracted from blood samples taken at various time points after cell infusion from mice transplanted with RNP-treated human CD34+ cells. Right panel: Detection of human cells in mouse peripheral blood samples. Each line corresponds to the level of gene editing detected in four individual mice transplanted with RNP-treated human CD34+ cells. Representative flow cytometry analysis of peripheral blood samples taken from RNP-treated mice 12 weeks after human HSC transplantation. Human CD45+ cells were distinguished from mouse CD45+ cells in the blood. Human lymphoid and myeloid lineages were detected within the human CD45+ cell gate (Figure 20E). Kinetics of hematopoietic reconstitution of NSG mice containing human CD45+ cells in recipients of RNP-treated and control untreated CD34+ cells from the same human HSC donor. Lower panel: Percentage of lymphoid and myeloid cells and erythroid progenitors detected within the human CD45+ cell gate in transplanted mice. [Figure 20D]Figure 20A shows the engraftment of Cas9 RNP gene-edited human CB CD34+ cells in an immune-deficient mouse model. (Figure 20A) Experimental schematic of RNP delivery to HSCs and transplantation and long-term tracking in NSG mice. (Figure 20B) Summary of the subtypes of editing events detected in an experiment in which fresh CD34+ cells were contacted with D10A nickase RNP and a gRNA pair (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) for use in HSC transplantation. (Figure 20C) Analysis of CFC hematopoietic activity of gene-edited HSCs (left), and DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for the detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from CFCs (right). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 20D) Left panel: Detection of indels by T7E1 assay in gDNA extracted from blood samples taken at various time points after cell infusion from mice transplanted with RNP-treated human CD34+ cells. Right panel: Detection of human cells in mouse peripheral blood samples. Each line corresponds to the level of gene editing detected in four individual mice transplanted with RNP-treated human CD34+ cells. Representative flow cytometry analysis of peripheral blood samples taken from RNP-treated mice 12 weeks after human HSC transplantation. Human CD45+ cells were distinguished from mouse CD45+ cells in the blood. Human lymphoid and myeloid lineages were detected within the human CD45+ cell gate (Figure 20E). Kinetics of hematopoietic reconstitution of NSG mice containing human CD45+ cells in recipients of RNP-treated and control untreated CD34+ cells from the same human HSC donor. Lower panel: Percentage of lymphoid and myeloid cells and erythroid progenitors detected within the human CD45+ cell gate in transplanted mice. [Figure 20E]Figure 20A shows the engraftment of Cas9 RNP gene-edited human CB CD34+ cells in an immune-deficient mouse model. (Figure 20A) Experimental schematic of RNP delivery to HSCs and transplantation and long-term tracking in NSG mice. (Figure 20B) Summary of the subtypes of editing events detected in an experiment in which fresh CD34+ cells were contacted with D10A nickase RNP and a gRNA pair (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) for use in HSC transplantation. (Figure 20C) Analysis of CFC hematopoietic activity of gene-edited HSCs (left), and DNA sequence analysis of HSC clones (erythroid and myeloid CFCs) for the detection of wild-type DNA sequences (no editing), monoallelic, or biallelic editing events at the HBB locus in gDNA from CFCs (right). E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythroid-macrophage-monocyte. (Figure 20D) Left panel: Detection of indels by T7E1 assay in gDNA extracted from blood samples taken at various time points after cell infusion from mice transplanted with RNP-treated human CD34+ cells. Right panel: Detection of human cells in mouse peripheral blood samples. Each line corresponds to the level of gene editing detected in four individual mice transplanted with RNP-treated human CD34+ cells. Representative flow cytometry analysis of peripheral blood samples taken from RNP-treated mice 12 weeks after human HSC transplantation. Human CD45+ cells were distinguished from mouse CD45+ cells in the blood. Human lymphoid and myeloid lineages were detected within the human CD45+ cell gate (Figure 20E). Kinetics of hematopoietic reconstitution of NSG mice containing human CD45+ cells in recipients of RNP-treated and control untreated CD34+ cells from the same human HSC donor. Lower panel: Percentage of lymphoid and myeloid cells and erythroid progenitors detected within the human CD45+ cell gate in transplanted mice. [Figure 21A]21A depicts the detection of gene-edited human HSCs in hematopoietic organs of transplanted NSG mice. (FIG. 21A) Representative flow cytometry gating schematic for detecting human CD45+ cells in mouse hematopoietic organs. Lower panel: Mean engraftment of human CD45+ cells in bone marrow and spleen of the indicated human HSC transplant groups. Lower right panel: Mean percentage of human CD34+ cells detected within the human CD45+ gate in mouse bone marrow and spleen. (FIG. 21B) Representative flow cytometry analysis for detecting human CD45+ and human CD34+ cells within the human CD45+ cell gate in bone marrow from transplanted mice before (upper panel) and after (lower panel) human cell isolation. (FIG. 21C) Gene editing detected by T7E1 analysis in hematopoietic organs (sorted human cells) and blood samples (unsorted) of mice transplanted with RNP-treated HSCs. [Figure 21B] 21A depicts the detection of gene-edited human HSCs in hematopoietic organs of transplanted NSG mice. (FIG. 21A) Representative flow cytometry gating schematic for detecting human CD45+ cells in mouse hematopoietic organs. Lower panel: Mean engraftment of human CD45+ cells in bone marrow and spleen of the indicated human HSC transplant groups. Lower right panel: Mean percentage of human CD34+ cells detected within the human CD45+ gate in mouse bone marrow and spleen. (FIG. 21B) Representative flow cytometry analysis for detecting human CD45+ and human CD34+ cells within the human CD45+ cell gate in bone marrow from transplanted mice before (upper panel) and after (lower panel) human cell isolation. (FIG. 21C) Gene editing detected by T7E1 analysis in hematopoietic organs (sorted human cells) and blood samples (unsorted) of mice transplanted with RNP-treated HSCs. [Figure 21C]21A depicts the detection of gene-edited human HSCs in hematopoietic organs of transplanted NSG mice. (FIG. 21A) Representative flow cytometry gating schematic for detecting human CD45+ cells in mouse hematopoietic organs. Lower panel: Mean engraftment of human CD45+ cells in bone marrow and spleen of the indicated human HSC transplant groups. Lower right panel: Mean percentage of human CD34+ cells detected within the human CD45+ gate in mouse bone marrow and spleen. (FIG. 21B) Representative flow cytometry analysis for detecting human CD45+ and human CD34+ cells within the human CD45+ cell gate in bone marrow from transplanted mice before (upper panel) and after (lower panel) human cell isolation. (FIG. 21C) Gene editing detected by T7E1 analysis in hematopoietic organs (sorted human cells) and blood samples (unsorted) of mice transplanted with RNP-treated HSCs. [Figure 22A] Figure 2 depicts the engraftment and gene editing frequency of human CD45+ cells in the peripheral blood, spleen, and bone marrow of immune-deficient NSG mice transplanted with human umbilical cord blood (CB) CD34+ HSCs electroporated with D10A Cas9 HBB-8 and HBB-15 gRNA RNP complexes (RNPs) after preconditioning with 20 mg / kg busulfan. Experiment 1 ("Expt1," 260,000 cells / mouse) panel depicts the long-term (4-month) engraftment and gene editing frequency corresponding to the human / mouse xenograft experiment depicted in Figure 21. [Figure 22B] Figure 2 depicts the engraftment and gene editing frequency of human CD45+ cells in the peripheral blood, spleen, and bone marrow of immune-deficient NSG mice transplanted with human umbilical cord blood (CB) CD34+ HSCs electroporated with D10A Cas9 HBB-8 and HBB-15 gRNA RNP complexes (RNPs) after preconditioning with 20 mg / kg busulfan. Experiment 1 ("Expt1," 260,000 cells / mouse) panel depicts the long-term (4-month) engraftment and gene editing frequency corresponding to the human / mouse xenograft experiment depicted in Figure 21. [Figure 22C]Figure 2 depicts the engraftment and gene editing frequency of human CD45+ cells in the peripheral blood, spleen, and bone marrow of immune-deficient NSG mice transplanted with human umbilical cord blood (CB) CD34+ HSCs electroporated with D10A Cas9 HBB-8 and HBB-15 gRNA RNP complexes (RNPs) after preconditioning with 20 mg / kg busulfan. Experiment 1 ("Expt1," 260,000 cells / mouse) panel depicts the long-term (4-month) engraftment and gene editing frequency corresponding to the human / mouse xenograft experiment depicted in Figure 21. [Figure 22D] Flow cytometry analysis of mouse peripheral blood samples from a representative animal of Expt1 showing the relative percentages of mouse and human hematopoietic CD45+ cell content in peripheral blood. Human lymphocyte subsets were analyzed within the total human blood (CD45+) cell gate. [Figure 22E] Gene editing frequencies as determined by DNA sequencing in blood, spleen, and bone marrow 16 weeks after HSC transplantation are shown. [Figure 22F] Gene editing frequencies as determined by DNA sequencing in blood, spleen, and bone marrow 16 weeks after HSC transplantation are shown. [Figure 22G] Gene editing frequencies as determined by DNA sequencing in blood, spleen, and bone marrow 16 weeks after HSC transplantation are shown. [Figure 22H] Figure 1 shows the frequency of specific types of gene editing events detected in sorted human cells from the bone marrow and spleen of a representative RNP-HSC transplant recipient from each group. Figure 2 shows the total gene editing frequency (determined by DNA sequencing analysis) and types of gene editing detected in enriched human cells from the spleen and bone marrow of mice transplanted with RNP-treated human CD34+ cells 4 months prior. Figure 3 shows the frequency of gene editing detected in the pre-transplant infusion (ex vivo) cell product and the frequency of gene editing detected in vivo in a representative transplant recipient. [Figure 22I]Figure 1 shows the frequency of specific types of gene editing events detected in sorted human cells from the bone marrow and spleen of a representative RNP-HSC transplant recipient from each group. Figure 2 shows the total gene editing frequency (determined by DNA sequencing analysis) and types of gene editing detected in enriched human cells from the spleen and bone marrow of mice transplanted with RNP-treated human CD34+ cells 4 months prior. Figure 3 shows the frequency of gene editing detected in the pre-transplant infusion (ex vivo) cell product and the frequency of gene editing detected in vivo in a representative transplant recipient. [Figure 23A] These panels depict the engraftment of human CD45+ cells in the peripheral blood, spleen, and bone marrow of immune-deficient NSG mice transplanted with human umbilical cord blood (CB) CD34+ HSCs that were preconditioned with 25 mg / kg busulfan, primed in medium containing cytokines, PGE2, and SR-1, electroporated with a complex of D10A Cas9 and HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) RNPs, and cultured for an additional 2 days in medium containing cytokines, PGE2, and SR-1. For Experiment 2 ("EXPT2"), each animal received 570,000 cells. All panels depict long-term (16 weeks or 4 months) engraftment. [Figure 23B] These panels depict the engraftment of human CD45+ cells in the peripheral blood, spleen, and bone marrow of immune-deficient NSG mice transplanted with human umbilical cord blood (CB) CD34+ HSCs that were preconditioned with 25 mg / kg busulfan, primed in medium containing cytokines, PGE2, and SR-1, electroporated with a complex of D10A Cas9 and HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) RNPs, and cultured for an additional 2 days in medium containing cytokines, PGE2, and SR-1. For Experiment 2 ("EXPT2"), each animal received 570,000 cells. All panels depict long-term (16 weeks or 4 months) engraftment. [Figure 23C]These panels depict the engraftment of human CD45+ cells in the peripheral blood, spleen, and bone marrow of immune-deficient NSG mice transplanted with human umbilical cord blood (CB) CD34+ HSCs that were preconditioned with 25 mg / kg busulfan, primed in medium containing cytokines, PGE2, and SR-1, electroporated with a complex of D10A Cas9 and HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) RNPs, and cultured for an additional 2 days in medium containing cytokines, PGE2, and SR-1. For Experiment 2 ("EXPT2"), each animal received 570,000 cells. All panels depict long-term (16 weeks or 4 months) engraftment. [Figure 23D] Flow cytometry analysis of mouse peripheral blood samples from a representative animal of EXPT2 showing the relative percentages of mouse and human hematopoietic CD45+ cell content in peripheral blood. Human lymphocyte subsets were analyzed within the total human blood (CD45+) cell gate. [Figure 23E] Gene editing frequencies as determined by DNA sequencing in blood, spleen, and bone marrow 16 weeks after HSC transplantation are shown. [Figure 23F] Gene editing frequencies as determined by DNA sequencing in blood, spleen, and bone marrow 16 weeks after HSC transplantation are shown. [Figure 23G] Gene editing frequencies as determined by DNA sequencing in blood, spleen, and bone marrow 16 weeks after HSC transplantation are shown. [Figure 23H]

[0023] Figure 1 shows the specific types of gene editing events detected in sorted human cells from the bone marrow and spleen of a representative RNP-HSC transplant recipient. The total gene editing frequency (determined by DNA sequencing analysis) and the types of editing detected in enriched human cells from the spleen and bone marrow of mice transplanted with RNP-treated human CD34+ cells 4 months prior. The frequency of gene editing detected in the pre-transplant infusion (ex vivo) cell product and the frequency of gene editing detected in vivo in a representative transplant recipient are shown. [Figure 23I]

[0023] Figure 1 shows the specific types of gene editing events detected in sorted human cells from the bone marrow and spleen of a representative RNP-HSC transplant recipient. The total gene editing frequency (determined by DNA sequencing analysis) and the types of editing detected in enriched human cells from the spleen and bone marrow of mice transplanted with RNP-treated human CD34+ cells 4 months prior. The frequency of gene editing detected in the pre-transplant infusion (ex vivo) cell product and the frequency of gene editing detected in vivo in a representative transplant recipient are shown. [Figure 24A] Flow cytometry data are shown for the proliferation of mouse bone marrow containing human CD34+ HSC, human myeloid (CD33+), and human erythroid (CD235+) cells 4 months after transplantation of RNP-treated human CD34+ cells. [Figure 24B] Gene editing detected in sorted human CD34+ HSCs, human CD33+ myeloid cells, and human erythroid CD235+ cells enriched from the bone marrow of transplanted mice, as determined by T7E1 analysis (left panel) and DNA sequencing (right panel), is shown. [Figure 25A] Figure 1 shows gene editing frequencies in 12-week-old human adult mobilized peripheral blood (mPB) CD34+ cells as determined by T7E1 endonuclease analysis and DNA sequencing. [Figure 25B] Figure 1 depicts short-term engraftment of human CD45+ lymphoid and myeloid cells differentiated from RNP-treated mPB CD34+ cells or untreated control mPB CD34+ cells in vivo 12 weeks after transplantation. [Figure 25C] Depict the kinetics of human CD45+ cell hematopoietic reconstitution with gene-edited mPB CD34+ cells. [Figure 25D] Representative flow cytometry analysis of peripheral blood from mice transplanted with RNP-treated or untreated control human mPB CD34+ cells is shown. [Figure 26A]Figure 26A shows gene editing and homologous recombination repair frequencies in human CB CD34+ cells after electroporation of D10A Cas9RNP complexed with HBB-8 and HBB-15 gRNA, with or without co-delivery of single-stranded oligonucleotide donors (ssODNs) with unmodified 5' and 3' ends (NM) or modified with a phosphorothioate group at both the 5' and 3' ends (Phx). Figure 26A shows gene editing frequencies (determined by T7E1 nuclease assay) in CB CD34+ cells after delivery of various amounts of ssODN. Figure 26B shows viability, as measured by fold change in the number of human CD34+ cells over time, relative to the time of electroporation with Cas9 RNP and two gRNAs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) and high and low amounts of unmodified and modified ssODN. Figure 26C depicts the total gene editing types and subsets of gene editing types determined by DNA sequencing analysis. Note that gene modification indicates the detection of ssODN sequences at the target site (HDR). Figure 26D shows the total HDR frequency (i.e., the sum of gene conversion and gene modification) achieved with or without co-delivery of ssODN into CB CD34+ cells. [Figure 26B]Figure 26A shows gene editing and homologous recombination repair frequencies in human CB CD34+ cells after electroporation of D10A Cas9RNP complexed with HBB-8 and HBB-15 gRNA, with or without co-delivery of single-stranded oligonucleotide donors (ssODNs) with unmodified 5' and 3' ends (NM) or modified with a phosphorothioate group at both the 5' and 3' ends (Phx). Figure 26A shows gene editing frequencies (determined by T7E1 nuclease assay) in CB CD34+ cells after delivery of various amounts of ssODN. Figure 26B shows viability, as measured by fold change in the number of human CD34+ cells over time, relative to the time of electroporation with Cas9 RNP and two gRNAs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) and high and low amounts of unmodified and modified ssODN. Figure 26C depicts the total gene editing types and subsets of gene editing types determined by DNA sequencing analysis. Note that gene modification indicates the detection of ssODN sequences at the target site (HDR). Figure 26D shows the total HDR frequency (i.e., the sum of gene conversion and gene modification) achieved with or without co-delivery of ssODN into CB CD34+ cells. [Figure 26C]Figure 26A shows gene editing and homologous recombination repair frequencies in human CB CD34+ cells after electroporation of D10A Cas9RNP complexed with HBB-8 and HBB-15 gRNA, with or without co-delivery of single-stranded oligonucleotide donors (ssODNs) with unmodified 5' and 3' ends (NM) or modified with a phosphorothioate group at both the 5' and 3' ends (Phx). Figure 26A shows gene editing frequencies (determined by T7E1 nuclease assay) in CB CD34+ cells after delivery of various amounts of ssODN. Figure 26B shows viability, as measured by fold change in the number of human CD34+ cells over time, relative to the time of electroporation with Cas9 RNP and two gRNAs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) and high and low amounts of unmodified and modified ssODN. Figure 26C depicts the total gene editing types and subsets of gene editing types determined by DNA sequencing analysis. Note that gene modification indicates the detection of ssODN sequences at the target site (HDR). Figure 26D shows the total HDR frequency (i.e., the sum of gene conversion and gene modification) achieved with or without co-delivery of ssODN into CB CD34+ cells. [Figure 26D]Figure 26A shows gene editing and homologous recombination repair frequencies in human CB CD34+ cells after electroporation of D10A Cas9RNP complexed with HBB-8 and HBB-15 gRNA, with or without co-delivery of single-stranded oligonucleotide donors (ssODNs) with unmodified 5' and 3' ends (NM) or modified with a phosphorothioate group at both the 5' and 3' ends (Phx). Figure 26A shows gene editing frequencies (determined by T7E1 nuclease assay) in CB CD34+ cells after delivery of various amounts of ssODN. Figure 26B shows viability, as measured by fold change in the number of human CD34+ cells over time, relative to the time of electroporation with Cas9 RNP and two gRNAs (HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) and high and low amounts of unmodified and modified ssODN. Figure 26C depicts the total gene editing types and subsets of gene editing types determined by DNA sequencing analysis. Note that gene modification indicates the detection of ssODN sequences at the target site (HDR). Figure 26D shows the total HDR frequency (i.e., the sum of gene conversion and gene modification) achieved with or without co-delivery of ssODN into CB CD34+ cells. [Figure 27A]Figures 27A and 27B show gene editing and HDR frequencies after co-delivery of D10A Cas9 RNP and two gRNAs (gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without Phx ssODN titrated down to less than 100 pmol per 200,000 cells. Figures 27A and 27B depict the frequency of gene editing events as determined by DNA sequencing analysis of HBB locus-specific PCR products. Figure 27C shows the in vitro differentiation or hematopoietic activity (i.e., colony-forming ability) of untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (two gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without ssODN. Figure 27D depicts the frequency of HDR events (determined by DNA sequencing analysis) in cells treated with or without 0, 50, 75, or 100 pmol of Phx-modified ssODN donor template. Figure 27E shows the ex vivo differentiation potential or hematopoietic activity (i.e., colony-forming ability) in untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (HBB-8 and HBB-15) with or without ssODN. CFC: colony-forming cells, M: macrophage colonies, GM: granulocyte-macrophage colonies, G: granulocyte colonies. [Figure 27B]Figures 27A and 27B show gene editing and HDR frequencies after co-delivery of D10A Cas9 RNP and two gRNAs (gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without Phx ssODN titrated down to less than 100 pmol per 200,000 cells. Figures 27A and 27B depict the frequency of gene editing events as determined by DNA sequencing analysis of HBB locus-specific PCR products. Figure 27C shows the in vitro differentiation or hematopoietic activity (i.e., colony-forming ability) of untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (two gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without ssODN. Figure 27D depicts the frequency of HDR events (determined by DNA sequencing analysis) in cells treated with or without 0, 50, 75, or 100 pmol of Phx-modified ssODN donor template. Figure 27E shows the ex vivo differentiation potential or hematopoietic activity (i.e., colony-forming ability) in untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (HBB-8 and HBB-15) with or without ssODN. CFC: colony-forming cells, M: macrophage colonies, GM: granulocyte-macrophage colonies, G: granulocyte colonies. [Figure 27C]Figures 27A and 27B show gene editing and HDR frequencies after co-delivery of D10A Cas9 RNP and two gRNAs (gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without Phx ssODN titrated down to less than 100 pmol per 200,000 cells. Figures 27A and 27B depict the frequency of gene editing events as determined by DNA sequencing analysis of HBB locus-specific PCR products. Figure 27C shows the in vitro differentiation or hematopoietic activity (i.e., colony-forming ability) of untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (two gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without ssODN. Figure 27D depicts the frequency of HDR events (determined by DNA sequencing analysis) in cells treated with or without 0, 50, 75, or 100 pmol of Phx-modified ssODN donor template. Figure 27E shows the ex vivo differentiation potential or hematopoietic activity (i.e., colony-forming ability) in untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (HBB-8 and HBB-15) with or without ssODN. CFC: colony-forming cells, M: macrophage colonies, GM: granulocyte-macrophage colonies, G: granulocyte colonies. [Figure 27D]Figures 27A and 27B show gene editing and HDR frequencies after co-delivery of D10A Cas9 RNP and two gRNAs (gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without Phx ssODN titrated down to less than 100 pmol per 200,000 cells. Figures 27A and 27B depict the frequency of gene editing events as determined by DNA sequencing analysis of HBB locus-specific PCR products. Figure 27C shows the in vitro differentiation or hematopoietic activity (i.e., colony-forming ability) of untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (two gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without ssODN. Figure 27D depicts the frequency of HDR events (determined by DNA sequencing analysis) in cells treated with or without 0, 50, 75, or 100 pmol of Phx-modified ssODN donor template. Figure 27E shows the ex vivo differentiation potential or hematopoietic activity (i.e., colony-forming ability) in untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (HBB-8 and HBB-15) with or without ssODN. CFC: colony-forming cells, M: macrophage colonies, GM: granulocyte-macrophage colonies, G: granulocyte colonies. [Figure 27E]Figures 27A and 27B show gene editing and HDR frequencies after co-delivery of D10A Cas9 RNP and two gRNAs (gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without Phx ssODN titrated down to less than 100 pmol per 200,000 cells. Figures 27A and 27B depict the frequency of gene editing events as determined by DNA sequencing analysis of HBB locus-specific PCR products. Figure 27C shows the in vitro differentiation or hematopoietic activity (i.e., colony-forming ability) of untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (two gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387)) with or without ssODN. Figure 27D depicts the frequency of HDR events (determined by DNA sequencing analysis) in cells treated with or without 0, 50, 75, or 100 pmol of Phx-modified ssODN donor template. Figure 27E shows the ex vivo differentiation potential or hematopoietic activity (i.e., colony-forming ability) in untreated controls and CD34+ cells electroporated with D10A RNP and two gRNAs (HBB-8 and HBB-15) with or without ssODN. CFC: colony-forming cells, M: macrophage colonies, GM: granulocyte-macrophage colonies, G: granulocyte colonies. [Figure 28] The frequency of total gene editing and the frequency of types of gene editing events (determined by DNA sequencing analysis) in two additional CB CD34+ cell donors and one adult mPBCD34+ cell donor after electroporation of D10A RNP complexed with HBB-8 and HBB-15 gRNAs and 100 pmol of Phx-modified ssODN per 200,000 cells are shown. [Figure 29A]Figure 29 depicts the effect of including a 5'-end modification (e.g., an ARCA cap) in gRNA with or without a 3'-end modification (e.g., a defined-length poly(A) tail, poly(T) tail, or poly(G) tail) on gene editing frequency in adult mPBCD34+ cells and their hematopoietic potential (e.g., CFCs). In the experiment depicted in Figure 29A, gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) were transcribed in vitro with the indicated 5'- and 3'-end modifications complexed with D10A Cas9 protein. Figure 29B shows gene editing frequency determined by DNA sequencing analysis. [Figure 29B] Figure 29 depicts the effect of including a 5'-end modification (e.g., an ARCA cap) in gRNA with or without a 3'-end modification (e.g., a defined-length poly(A) tail, poly(T) tail, or poly(G) tail) on gene editing frequency in adult mPBCD34+ cells and their hematopoietic potential (e.g., CFCs). In the experiment depicted in Figure 29A, gRNAs HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387) were transcribed in vitro with the indicated 5'- and 3'-end modifications complexed with D10A Cas9 protein. Figure 29B shows gene editing frequency determined by DNA sequencing analysis. [Figure 29C] Using a dual nickase strategy, we delineated the CFC capacity of CB CD34+ cells after electroporation with D10A Cas9 RNP targeting the HBB locus, where both HBB-8 and HBB-15 were modified at the 5' and / or 3' ends with the indicated modifications: E: erythrocyte, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte. [Figure 30A]A dual-nickase strategy was used to depict the gene editing frequency and CFC capacity of CB CD34+ cells after electroporation with D10A Cas9 RNP targeting the HBB locus, where two gRNAs, HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387), are both 5'-modified and have the indicated 3'-end modifications: E: erythrocyte, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte. [Figure 30B] A dual-nickase strategy was used to depict the gene editing frequency and CFC capacity of CB CD34+ cells after electroporation with D10A Cas9 RNP targeting the HBB locus, where two gRNAs, HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387), are both 5'-modified and have the indicated 3'-end modifications: E: erythrocyte, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte. [Figure 30C] A dual-nickase strategy was used to depict the gene editing frequency and CFC capacity of CB CD34+ cells after electroporation with D10A Cas9 RNP targeting the HBB locus, where two gRNAs, HBB-8 (SEQ ID NO: 388) and HBB-15 (SEQ ID NO: 387), are both 5'-modified and have the indicated 3'-end modifications: E: erythrocyte, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte. DETAILED DESCRIPTION OF THE INVENTION

[0098] The methods and compositions described herein enhance CRISPR / Cas9-mediated gene editing in stem cells, such as HSCs. Exposing stem cells to foreign molecules, such as CRISPR / Cas9 system components (e.g., gRNA molecules, Cas9 molecules, and / or template nucleic acids), stresses stem cells and induces innate immune responses, which trigger, for example, programmed cell death or stem cell differentiation. By using the methods and systems described herein, the stress response and innate immune response signaling events that can ultimately cause programmed cell death or stem cell differentiation in stem cells after exposure to foreign CRISPR / Cas9 components are reduced or neutralized. Specifically, by temporarily exposing stem cells to a stem cell viability enhancer (e.g., for a period of less than 120 hours) before and / or after contacting the stem cells with one or more CRISPR / Cas9 components, the innate immune response of stem cells to foreign CRISPR / Cas9 components is reduced or blocked, thereby significantly increasing stem cell viability. Additionally, brief, transient treatment of stem cells with stem cell viability enhancers (e.g., small molecules) also results in increased stem cell multipotency and self-renewal capacity. Furthermore, the methods and compositions disclosed herein also increase stem cell engraftment in target tissues upon introduction of the modified stem cells into a subject.

[0099] The unexpected benefit of transient exposure of stem cells to the stem cell viability agents disclosed herein is surprising given that these agents are typically used as stem cell expansion agents, e.g., to enhance stem cell proliferation and increase their numbers several-fold (e.g., at least five-fold), and that stem cell expansion benefits require prolonged exposure to the agent (e.g., exposure in cell culture for more than one week). For example, to demonstrate a significant increase in stem cell numbers in culture, Boitono et al. cultured stem cells in the presence of SR1 for three weeks (see Boitono et al. 2010), and Gu et al. demonstrated that stem cells cultured with SR1 for seven days lost lymphoid reconstitution capacity compared to untreated cells, indicating that SR1 was not effective in expanding viable stem cells. Such loss of stem cell multipotency was also observed by Carlin et al. (Cytotherapy, 2013), where co-culture of stem cells with another AhR antagonist (AhRA and dimethoxyflavone, DMF) for 14 days resulted in a loss of in vitro lymphoid differentiation potential.

[0100] Thus, the methods and compositions described herein are particularly advantageous for optimizing editing of target nucleic acid sequences that result in viable stem cells, advancing the field of stem cell therapy in a variety of clinical applications.

[0101] definition The term "viability," as used herein, refers to the ability of a stem cell to survive, maintain itself, and / or regain its potential after exposure to, for example, a CRISPR / Cas9 system. In one embodiment, the viable stem cell can be maintained in cell culture.

[0102] The terms "expand" or "expansion," as used herein, refer to the growth of stem cells (e.g., CD34 cells) over a period of at least 168 hours (e.g., 7 days) and under conditions that result in an increase in cell number of more than five-fold the initial cell number by the end of the at least 168 hour (e.g., 7 day) period. + This refers to culturing HSC cells.

[0103] The terms "maintain," "maintain," "maintaining," or "maintained," as used herein with reference to cells in culture, refer to culturing cells for a period of time and under conditions that support cell viability, wherein the increase in cell number during or by the end of the period is less than 5-fold relative to the initial cell number. In one embodiment, the increase in cell number is less than 4-fold the initial cell number. In one embodiment, the increase in cell number is less than 3-fold the initial cell number. In one embodiment, the increase in cell number is less than 2-fold the initial cell number. In one embodiment, the increase in cell number is less than 1.6-fold the initial cell number. In one embodiment, the fold change in cell number is more than 1-fold but less than 2-fold the initial cell number. In another embodiment, the period is less than about 72 hours. In one embodiment, the period is less than about 48 hours. In another embodiment, the period is less than about 24 hours. For example, in one embodiment, the term maintenance refers to culturing stem cells for a period of about 72 hours under conditions where the cell number increases less than 5-fold during a period of about 96 hours, or by the end of the period of about 72 hours. In another embodiment, the term maintenance refers to culturing stem cells for a period of about 72 hours under conditions where the cell number increases less than 4-fold during a period of about 72 hours, or by the end of the period of about 72 hours.

[0104] The term "cell viability enhancer" or "stem cell viability enhancer," as used herein, refers to a compound (e.g., a nucleotide, protein, or small molecule) that can increase the viability of a stem cell following exposure (e.g., electroporation) to a CRISPR / Cas9 system (e.g., a Cas9 system described herein) compared to the viability of the same type of stem cell following exposure to the Cas9 system without treatment with the stem cell viability enhancer. In some embodiments, the stem cell viability enhancer is a protein. In some embodiments, the stem cell viability enhancer is a peptide. In some embodiments, the stem cell viability enhancer is a small molecule. In some embodiments, the stem cell viability enhancer is a nucleic acid (e.g., siRNA, shRNA, mRNA, DNA, RNA). In some embodiments, the stem cell viability enhancer is cell membrane permeable. In some embodiments, the stem cell viability enhancer is not cell membrane permeable. In some embodiments, the stem cell viability enhancer is nuclear membrane permeable. In some embodiments, the stem cell viability enhancer is not nuclear membrane permeable. In some embodiments, the stem cell viability enhancer is an aryl hydrocarbon receptor antagonist. In some embodiments, the stem cell viability enhancer is a pyrimidoindole derivative. In some embodiments, the stem cell viability enhancer is an innate immune response antagonist. In one embodiment, the stem cell viability enhancer is MG132. In another embodiment, the stem cell viability enhancer is SB431542. In another embodiment, the stem cell viability enhancer is UM171. In another embodiment, the stem cell viability enhancer is UM729. In another embodiment, the stem cell viability enhancer is 16,16-dimethyl prostaglandin E2 (dmPGE2).

[0105] The term "innate immune response agonist," as used herein, refers to a molecule that inhibits a stem cell's innate immune response (e.g., in response to contact of a stem cell with one or more components of a Cas9 system (e.g., by electroporation)). In some embodiments, the stem cell viability enhancer inhibits signaling events necessary for a stem cell's innate immune response (e.g., in response to contact of a stem cell with one or more components of a Cas9 system (e.g., by electroporation)) to occur. In some embodiments, the stem cell viability enhancer inhibits cell death (e.g., programmed cell death) of a stem cell. In some embodiments, the stem cell viability enhancer inhibits programmed cell death of a stem cell (e.g., in response to contact of a stem cell with one or more components of a Cas9 system). In some embodiments, the stem cell viability enhancer inhibits signaling events necessary for a cell to undergo programmed cell death signaling events (e.g., in response to contact of a stem cell with one or more components of a Cas9 system). In some embodiments, the stem cell viability enhancer inhibits senescence in stem cells. In some embodiments, the stem cell viability enhancer inhibits stem cell differentiation. In one embodiment, the stem cell viability enhancer inhibits apoptosis of stem cells. In one embodiment, the stem cell viability enhancer inhibits autophagy of stem cells. In some embodiments, the stem cell viability enhancer increases the frequency of stem cell implantation into the target tissue compared to the frequency of stem cell implantation into the target tissue without treatment with the stem cell viability enhancer.

[0106] Examples of innate immune response antagonists are known in the art. For example, in one embodiment, the innate immune response antagonist is cyclosporin A. In another embodiment, the innate immune response antagonist is dexamethasone. In another embodiment, the innate immune response antagonist is resveratrol. In another embodiment, the innate immune response antagonist is a MyD88 inhibitory peptide. In another embodiment, the innate immune response antagonist is an RNAi agent targeting Myd88. In another embodiment, the innate immune response antagonist is B18R recombinant protein. In another embodiment, the innate immune response antagonist is a glucocorticoid. In another embodiment, the innate immune response antagonist is OxPAPC. In another embodiment, the innate immune response antagonist is a TLR antagonist. In another embodiment, the innate immune response antagonist is rapamycin. In another embodiment, the innate immune response antagonist is BX795. In another embodiment, the innate immune response antagonist is an RLR inhibitor.

[0107] The term "aryl hydrocarbon receptor antagonist," as used herein, refers to a molecule that inhibits the activity of the aryl hydrocarbon receptor. Such molecules are known in the art. In one embodiment, the AhR antagonist is StemRegenin-1 (SR1), also known as 4-[2-[[2-benzo[b]thien-3-yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]-phenol. In another embodiment, the AhR antagonist is AhRA. In another embodiment, the AhR antagonist is dimethoxyflavote. In another embodiment, the AhR antagonist is 6,2',4'-trimethoxyflavone. In another embodiment, the AhR antagonist is LGC0006. In another embodiment, the AhR antagonist is α-naphthoflavone. In yet another embodiment, the AhR antagonist is CH-223191.

[0108] The terms "transplant" and "transplantation," as used herein, refer to the process of transferring cells (e.g., modified cells described herein) into and / or onto a host subject. In one embodiment, transplantation involves introducing stem cells into the patient's bloodstream or bone marrow. In another embodiment, transplantation involves introducing stem cells into a solid tissue or tumor of the patient. In some embodiments, after transplantation, the transferred cells engraft into the subject's tissue.

[0109] As used herein, the term "engraft" refers to the process of incorporating cells into a target tissue through contact with the tissue. As used herein, the term "target tissue" refers to any group of similar cells and their surrounding extracellular material. In some embodiments, target tissues include, but are not limited to, epithelium, connective tissue (e.g., blood, bone, and cartilage), muscle tissue, and nervous tissue. In some embodiments, target tissues include, but are not limited to, peripheral blood, bone marrow, blood vessels, spleen, heart, liver, kidney, and skin.

[0110] As used herein, the terms "differentiate" and "differentiation," when used to describe cellular processes, refer to the acquisition or possession of one or more characteristics or functions that differ from those of the cell prior to transplantation. In some embodiments, the term "differentiation" includes the developmental process of lineage commitment. "Lineage" refers to the pathway of cell development in which precursor, or "progenitor," cells undergo stepwise physiological changes to become specialized cell types with specific functions (e.g., nerve cells, muscle cells, immune cells, or endothelial cells). Differentiation typically occurs in multiple stages, whereby primitive stem cells pass through several stages in which they lose the ability to self-renew and generate various cell types and become increasingly specialized until they reach full maturity, also referred to as "terminal differentiation." A "terminally differentiated cell" is a cell that has committed to a specific lineage and reached the final stage of differentiation (i.e., a fully mature cell).

[0111] "Progenitor cell," as used herein, refers to a lineage cell that is derived from a stem cell and retains mitotic capacity and multipotency (e.g., can differentiate or develop into two or more, if not all, mature lineage cell types).

[0112] "Hematopoiesis" or "hemopoiesis," as used herein, refers to the formation and development of various blood cells (e.g., red blood cells, megakaryocytes, myeloid cells (e.g., monocytes, macrophages, and neutrophils), and lymphocytes) and other formative elements in the body (e.g., in the bone marrow).

[0113] The terms "CRISPR / Cas9 system," "CRISPR / Cas9 system," or "Cas9 system," as used herein, refer to a system capable of modifying a target nucleic acid through one of many DNA repair pathways. In certain embodiments, the Cas9 system described herein promotes repair of the target nucleic acid via the HDR pathway. In some embodiments, the Cas9 system comprises a gRNA molecule and a Cas9 molecule. In some embodiments, the Cas9 system further comprises a second gRNA molecule. In yet other embodiments, the Cas9 system comprises a gRNA molecule, a Cas9 molecule, and a second gRNA molecule. In some embodiments, the Cas9 system comprises a gRNA molecule, two Cas9 molecules, and a second gRNA molecule. In some embodiments, the Cas9 system comprises a first gRNA molecule, a second gRNA molecule, a first Cas9 molecule, and a second Cas9 molecule. In exemplary embodiments, the Cas9 system further comprises a template nucleic acid, e.g., a single-stranded oligonucleotide. In one embodiment, the "gene editing system" or "stem cell gene editing system" is a kit that includes components of a CRISPR / Cas9 system.

[0114] As used herein, a "Cas9 molecule" refers to a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A "Cas9 polypeptide" is a polypeptide that can interact with a gRNA molecule and, in conjunction with the gRNA molecule, localize to a site containing a targeting domain, in certain embodiments, a PAM sequence. Cas9 molecules include both native Cas9 molecules and Cas9 molecules, as well as engineered, modified, or repaired Cas9 molecules, or Cas9 polypeptides that differ by at least one amino acid residue from a reference sequence, e.g., the most similar native Cas9 molecule. (The terms modified, engineered, or repaired, as used herein, simply refer to differences from a reference or native sequence and do not limit a specific process or source.) A Cas9 molecule may be a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide. A Cas9 molecule may be a nuclease (an enzyme that cleaves both strands of a double-stranded nucleic acid), a nickase (an enzyme that cleaves one strand of a double-stranded nucleic acid), or an enzymatically inactive (or inactive) Cas9 molecule. Cas9 molecules that have nuclease or nickase activity are referred to as "enzymatically active Cas9 molecules" ("eaCas9" molecules). Cas9 molecules that lack the ability to cleave target nucleic acids are referred to as "enzymatically inactive Cas9 molecules" ("eiCas9" molecules).

[0115] The term "gRNA molecule" or "gRNA," as used herein, refers to a guide RNA capable of targeting a Cas9 molecule to a target nucleic acid. In one embodiment, the term "gRNA molecule" refers to a guide ribonucleic acid. In another embodiment, the term "gRNA molecule" refers to a nucleic acid encoding a gRNA. In one embodiment, the gRNA molecule is non-naturally occurring. In one embodiment, the gRNA molecule is a synthetic gRNA molecule.

[0116] As used herein, a "modified gRNA molecule" or "modified gRNA" refers to a gRNA molecule that has an improved half-life after being introduced into a cell compared to an unmodified gRNA molecule after being introduced into a cell. In one embodiment, the modified gRNA molecule does not activate an innate immune response in a cell when the cell is exposed to the gRNA molecule (e.g., electroporated). In one embodiment, the modified gRNA molecule activates a reduced innate immune response in a cell when the cell is exposed to the gRNA molecule compared to the innate immune response of the cell when the same type of cell is exposed to the unmodified gRNA molecule. In another embodiment, the modified gRNA molecule does not activate a programmed cell death pathway (e.g., an apoptotic cell death pathway, a necrotic cell death pathway (e.g., a necrotic cell death pathway), an autophagy cell death pathway, an aponecrotic cell death pathway, a ferroptosis cell death pathway, an eryptosis cell death pathway, an aponecrotic cell death pathway, or an anoikis cell death pathway) in a cell when the cell is exposed to the gRNA molecule. In some embodiments, the modified gRNA molecule does not activate a caspase-dependent cell death pathway. In other embodiments, the modified gRNA molecule does not activate a caspase-independent cell death pathway.

[0117] In one embodiment, the modified gRNA molecule comprises a 5'-end modification. In one embodiment, the 5'-end modification is selected from the group consisting of: G(5')ppp(5')G cap analog, m7G(5')ppp(5')G cap analog, or 3'-O-Me-m7G(5')ppp(5')G anti-reverse cap analog (ARCA). In one embodiment, the 5'-end modification is a phosphorothioate modification. In one embodiment, the gRNA molecule comprises a 3'-end modification. In one embodiment, the 3'-end modification is a polyadenine tail. In one embodiment, the 3'-end modification is a phosphorothioate modification.

[0118] As used herein, "template nucleic acid" refers to a nucleic acid sequence that can be used together with a Cas9 molecule and a gRNA molecule to alter the structure of a target location. In one embodiment, the target nucleic acid is modified to have part or all of the sequence of the template nucleic acid, typically at or near the cleavage site. In one embodiment, the template nucleic acid is single-stranded. In another embodiment, the template nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, e.g., double-stranded DNA. In another embodiment, the template nucleic acid is single-stranded DNA. In one embodiment, the template nucleic acid is RNA, e.g., double-stranded RNA or single-stranded RNA. In one embodiment, the template nucleic acid is encoded in the same vector backbone as the Cas9 and gRNA, e.g., an AAV genome, plasmid DNA. In one embodiment, the template nucleic acid is excised from the vector backbone in vivo, e.g., it is flanked by gRNA recognition sequences. In one embodiment, the template DNA is in an ILDV. In one embodiment, the template nucleic acid is an exogenous nucleic acid sequence. In another embodiment, the template nucleic acid sequence is an endogenous nucleic acid sequence, e.g., an endogenous homology region. In one embodiment, the template nucleic acid is a single-stranded oligonucleotide corresponding to the plus strand of the nucleic acid sequence. In another embodiment, the template nucleic acid is a single-stranded oligonucleotide corresponding to the minus strand of the nucleic acid sequence.

[0119] As used herein, a "modified template nucleic acid" refers to a template nucleic acid, e.g., a single-stranded oligonucleotide molecule that serves as a template, that has an improved half-life after being introduced into a cell compared to the unmodified template nucleic acid after being introduced into the cell. In one embodiment, the modified template nucleic acid does not activate an innate immune response in a cell when exposed to the modified template nucleic acid (e.g., electroporated). In one embodiment, the modified template nucleic acid activates a reduced innate immune response in a cell when the cell is exposed to a gRNA molecule compared to the innate immune response of the cell when the same type of cell is exposed to the unmodified template nucleic acid. In another embodiment, the modified template nucleic acid does not activate a programmed cell death pathway (e.g., an apoptotic cell death pathway, a necrotic cell death pathway (e.g., a necrotic cell death pathway), an autophagy cell death pathway, an aponecrotic cell death pathway, a ferroptosis cell death pathway, an eryptosis cell death pathway, an aponecrotic cell death pathway, or an anoikis cell death pathway) in a cell when the cell is exposed to the modified template nucleic acid. In some embodiments, the modified template nucleic acid does not activate a caspase-dependent cell death pathway, hi other embodiments, the modified template nucleic acid does not activate a caspase-independent cell death pathway.

[0120] In one embodiment, the modified template nucleic acid is a single-stranded oligodeoxynucleotide (ssODN). In one embodiment, the ssODN comprises a 5' phosphorothioate modification. In one embodiment, the ssODN comprises a 3' phosphorothioate modification. In one embodiment, the ssODN comprises a 5' phosphorothioate modification and a 3' phosphorothioate modification.

[0121] "Target mutation position," as used herein, refers to a target position within a gene (e.g., a gene described herein) that, when mutated, results in a mutant protein and can cause a disease, e.g., a disease described herein.

[0122] "Target knockout position," as used herein, refers to a position within a gene (e.g., a gene described herein) that, when modified, results in a disease or amelioration of symptoms of a disease described herein.

[0123] "Target knockdown location," as used herein, refers to a location within a gene (e.g., a gene described herein) that, when targeted, e.g., by a Cas9 molecule described herein, results in the reduction or elimination of expression of a functional gene product.

[0124] "Targeted knock-in site," as used herein, refers to a sequence that, when modified by the insertion of a sequence of a gene (e.g., a gene described herein), results in optimization of gene activity, e.g., by providing a gene sequence that encodes a protein with wild-type activity.

[0125] "Target position," as used herein, refers to either a targeted mutation position, a targeted knockout position, a targeted knockdown position, or a targeted knockin position, as described herein.

[0126] " HDR " or " homology-directed repair " as used herein refers to the process of repairing DNA damage using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., template nucleic acid).Standard HDR typically operates when there is significant excision in double-strand break, and forms at least a single-stranded portion of DNA.In normal cells, HDR typically includes a series of steps, such as recognition of break, stabilization of break, excision, stabilization of single-stranded DNA, formation of DNA crossover intermediate, division of crossover intermediate, and joining.This process requires RAD51 and BRCA2, and homologous nucleic acid is typically double-stranded.

[0127] " Alt-HDR " or " alternative HDR " or alternative homology recombination repair, as used herein, refers to the process of using homologous nucleic acid (e.g., endogenous homologous sequence, e.g., sister chromatid, or exogenous nucleic acid, e.g., template nucleic acid) to repair DNA damage.Alt-HDR differs from standard HDR in that this process uses a different pathway from standard HDR and can be inhibited by standard HDR mediators RAD51 and BRCA2.Alt-HDR also uses single-stranded or nick homologous nucleic acid to repair breakage.

[0128] Unless otherwise specified, the term "HDR" as used herein encompasses HDR and alt-HDR.

[0129] As used herein, "gene conversion" refers to the process of repairing DNA damage by homologous recombination (HDR) using an endogenous nucleic acid, such as a sister chromatid or a plasmid, as a template nucleic acid. Without wishing to be bound by theory, in some embodiments, BRCA1, BRCA2, and / or RAD51 are thought to be involved in gene conversion. In some embodiments, the endogenous nucleic acid is a nucleic acid sequence that has homology, for example, significant homology, with a fragment of DNA near the site of DNA damage or mutation. In some embodiments, the template is not an exogenous nucleic acid.

[0130] "Gene correction," as used herein, refers to the process of repairing DNA damage by homologous recombination using an exogenous nucleic acid, such as a donor template nucleic acid. In some embodiments, the exogenous nucleic acid is single-stranded. In some embodiments, the exogenous nucleic acid is double-stranded.

[0131] As used herein, "genetic modification" refers to the process of editing nucleic acids using the CRISPR / Cas9 system described herein. In certain embodiments, genetic modification comprises gene correction. In certain embodiments, genetic modification comprises gene conversion.

[0132] "Non-homologous end joining" or "NHEJ," as used herein, refers to joining-mediated repair and / or non-template-mediated repair, including canonical NHEJ (cNHEJ), alternative NHEJ (altNHEJ), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0133] "Domain," as used herein, is used to describe a segment of a protein or nucleic acid. Unless otherwise specified, a domain does not need to have any particular functional properties.

[0134] "Modulator," as used herein, refers to an entity, such as, for example, an agent, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a molecule or gene sequence of interest. In one embodiment, modulation includes cleavage, e.g., breaking a covalent or non-covalent bond, or formation of a covalent or non-covalent bond, e.g., attaching a moiety to the molecule of interest. In one embodiment, a modulator alters the three-dimensional, secondary, tertiary, or quaternary structure of the molecule of interest. A modulator may increase, decrease, initiate, or eliminate an activity of interest.

[0135] "Large molecule," as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologics, and carbohydrates.

[0136] A "polypeptide," as used herein, refers to a polymer of amino acids having fewer than 100 amino acid residues. In one embodiment, it has fewer than 50, 20, or 10 amino acid residues.

[0137] As used herein, a "reference molecule," e.g., a reference Cas9 molecule or a reference gRNA, refers to a molecule to which a molecule of interest, e.g., a subject Cas9 molecule or a subject gRNA molecule, e.g., a modified or candidate Cas9 molecule, is compared. For example, a Cas9 molecule may be characterized as having 10% or less of the nuclease activity of the reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring, unmodified Cas9 molecules, e.g., naturally occurring Cas9 molecules, e.g., S. pyogenes, S. aureus, or S. thermophilus Cas9 molecules. In one embodiment, a reference Cas9 molecule is a naturally occurring Cas9 molecule that has the closest sequence identity or homology to the Cas9 molecule to which it is being compared. In one embodiment, a reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, e.g., a parental form to which changes, e.g., mutations, have been made.

[0138] "Substituted" or "replaced," as used herein in reference to molecular modification, does not necessarily imply a limitation on the process, but merely that a replacement entity is present.

[0139] "Small molecule," as used herein, refers to a compound having a molecular weight of less than about 2 kD, for example, less than about 2 kD, less than about 1.5 kD, less than about 1 kD, or less than about 0.75 kD.

[0140] "Subject," as used herein, can mean either a human or a non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In one embodiment, the subject is a human. In another embodiment, the subject is poultry.

[0141] "Treat," "treating," and "treatment," as used herein, refer to the treatment of a disease in a mammal, e.g., a human, including, for example, (a) inhibiting the disease, i.e., arresting or preventing its progression; (b) palliating the disease, i.e., causing regression of the disease state; and (c) curing the disease.

[0142] "Prevent," "preventing," and "prevention," as used herein, refer to the prevention of disease in a mammal, e.g., a human, and include (a) avoiding or eliminating the disease; (2) affecting predisposition to the disease, e.g., preventing at least one symptom of the disease or delaying the onset of at least one symptom of the disease.

[0143] "X," when used herein in reference to an amino acid sequence, refers to any amino acid (eg, any of the 20 naturally occurring amino acids), unless otherwise specified.

[0144] I. Stem Cell Optimization Stem cells, for example, the stem cells described herein, can be optimized or manipulated, for example, in vitro or in vivo.Without wishing to be bound by theory, in one embodiment, it is believed that the optimization or manipulation of stem cells can maintain, sustain, or regulate cells for CRISPR / Cas-mediated gene editing or regulation.For example, the optimization or manipulation of stem cells, for example, hematopoietic stem / progenitor cells (HSCs), can maintain cytocompatibility, functionality, self-renewal, engraftment ability, or prevent cell death via, for example, cell death mechanisms, such as autophagy, apoptosis, necrosis, aponecrosis, ferroptosis, eryptosis, anoikis, or cellular senescence.

[0145] In certain embodiments, but without wishing to be bound by theory, it is believed that contacting cells with stem cell viability enhancer can desirably promote cytocompatibility, functionality, self-renewal, engraftment ability, or prevent cell death.Stem cell viability enhancer will be described in more detail below.For example, contacting cells with CRISPR / Cas9 components, such as Cas9 molecules, gRNA molecules, or both, and optionally template nucleic acid, can trigger one or more cellular events (e.g., signal transduction events) that cause programmed cell death and / or innate immune response in stem cells.Therefore, by contacting stem cells with the stem cell viability enhancer described herein, stem cells can be easily manipulated using the CRISPR / Cas9 system.

[0146] Stem cells can be optimized or manipulated before, during, or after contact with CRISPR / Cas9 components, e.g., Cas9 molecules, gRNA molecules, or both, and, optionally, donor template nucleic acid. In one embodiment, stem cells are optimized or manipulated before contact with CRISPR / Cas9 components. In one embodiment, stem cells are optimized or manipulated during contact with CRISPR / Cas9 components. In one embodiment, stem cells are optimized or manipulated after contact with CRISPR / Cas9 components. In one embodiment, stem cells are optimized or manipulated before and during contact with CRISPR / Cas9 components. In one embodiment, stem cells are optimized or manipulated during and after contact with CRISPR / Cas9 components. In one embodiment, stem cells are optimized or manipulated before and after contact with CRISPR / Cas9 components. In one embodiment, stem cells are optimized or manipulated before, during, and after contact with CRISPR / Cas9 components.

[0147] Several different optimization or manipulation steps can be applied sequentially, e.g., at specific time intervals relative to contacting the CRISPR / Cas9 components, e.g., Cas9 molecules, gRNA molecules, or both, and optionally with the donor template nucleic acid. Also, several different optimization or manipulation steps can be applied simultaneously, e.g., at specific time intervals relative to contacting the CRISPR / Cas9 components, e.g., Cas9 molecules, gRNA molecules, or both, and optionally with the donor template nucleic acid.

[0148] For example, stem cells can be optimized or engineered by contacting them with one or more stem cell viability enhancers (e.g., cell agonists). As another example, stem cells can be optimized or engineered to contain one or more transgenes. The transgenes can be integrated into specific loci within the stem cell genome, for example, by CRISPR / Cas9-associated mechanisms. In one embodiment, the transgene can provide a safety switch that can allow for controlled enrichment and / or purification of modified cells prior to transplantation. In one embodiment, the transgene can also allow for in vivo expansion of modified cells if engrafted cells are not detected sufficiently, or for in vivo removal of modified cells if they are dysfunctional or undergo leukemic transformation. As yet another example, stem cells can be optimized or engineered by contacting them with one or more eiCas9 molecules, e.g., fused to a transcriptional repressor or activator.

[0149] Stem Cell Viability Enhancer Stem cells described herein can be contacted with one or more stem cell viability enhancers (e.g., an aryl hydrocarbon receptor (AhR) antagonist, an innate immune response antagonist, or other stem cell viability enhancers described herein), for example, to promote cell survival and prevent cell death that may occur in response to contact of the CRISPR / Cas9 components with the cell (e.g., in response to a signaling event (e.g., an innate immune response) or a gene editing event triggered by the CRISPR / Cas9 components).

[0150] In one embodiment, the stem cell viability enhancer is an AhR antagonist. In one embodiment, the stem cell viability enhancer is a pyrimido-[4,5-b]-indole derivative. In one embodiment, the stem cell viability enhancer is an innate immune response antagonist. In one embodiment, the stem cell viability enhancer is prostaglandin E2. In one embodiment, the stem cell viability enhancer is an NFκB inhibitor. In one embodiment, the stem cell viability enhancer is an mTOR inhibitor. In one embodiment, the stem cell viability enhancer is a MyD88 inhibitor. In one embodiment, the stem cell viability enhancer is a TGF-β inhibitor. In one embodiment, the stem cell viability enhancer is a Toll-like receptor (TLR) inhibitor. In one embodiment, the stem cell viability enhancer is a reactive nitrogen and oxygen species inhibitor. In one embodiment, the stem cell viability enhancer is a proteosome inhibitor. In one embodiment, the stem cell viability enhancer is a histone acetyltransferase inhibitor. In one embodiment, the stem cell viability enhancer is a c-MPL agonist. In one embodiment, the stem cell viability enhancer is an Ndr kinase modulator.

[0151] Previously, some stem cell viability enhancers have been found to help expand stem cell population.However, the method described herein does not need to contact stem cell for a long enough period to promote stem cell expansion.On the contrary, the method described herein only needs to contact stem cell with stem cell viability enhancer before and / or after exposure to CRISPR / Cas9 components for a long enough period to enhance stem cell viability, for example, inhibit innate immune response and / or innate immune response.

[0152] For example, stem cells can be cultured in StemSpan SFEM containing SCF, TPO, FL, and optionally one or more stem cell viability enhancers 1-120 hours before and up to 96 hours after contact with CRISPR / Cas9 components (e.g., Cas9 molecules, gRNA molecules, or both, and optionally, donor template nucleic acid). To enhance survival, stem cells can be contacted with more of the following stem cell viability enhancers after contact with CRISPR / Cas9 components to enhance survival and prevent cell death that can occur upon cellular perturbation by exposure to exogenous or foreign / unfamiliar nucleic acids, proteins, or viral particles containing CRISPR / Cas9 components. Without wishing to be bound by theory, brief pre-treatment (e.g., before contact with CRISPR / Cas9 components) and post-treatment (e.g., after contact with CRISPR / Cas9 components) of stem cells with stem cell viability enhancers can promote stem cell survival and maintenance and prevent stem cell senescence and cell death (e.g., via programmed cell death pathways).

[0153] In one embodiment, the stem cell is contacted with the stem cell viability enhancer before contacting the stem cell with the CRISPR / Cas9 components. In one embodiment, the stem cell is contacted with the stem cell viability enhancer after contacting the stem cell with the CRISPR / Cas9 components. In one embodiment, the target stem cell is contacted with the stem cell viability enhancer before contacting the cell with the CRISPR / Cas9 components and after contacting the cell with the CRISPR / Cas9 components.

[0154] In one embodiment, the stem cells are contacted with the stem cell viability enhancer for a period of less than 72 hours after contacting the stem cells with the CRISPR / Cas9 components. In one embodiment, the period of less than 72 hours is about 48 hours. In one embodiment, the period of less than 72 hours is about 48 hours plus or minus about 12 hours. In one embodiment, the period of less than 72 hours is about 24 hours. In one embodiment, the period of less than 72 hours is about 48 hours or less. In one embodiment, the period of less than 72 hours is about 24 hours or less. In certain embodiments, the stem cells are cryopreserved within about 48 hours after the end of the period of less than 72 hours. In certain embodiments, the stem cells are cryopreserved within about 24 hours after the end of the period of less than 72 hours. In certain embodiments, the stem cells are transferred into a subject (e.g., a human subject) within about 48 hours after the end of the period of less than 72 hours. In certain embodiments, the stem cells are transferred into a subject (e.g., a human subject) within about 24 hours after the end of the period of less than 72 hours.

[0155] In one embodiment, the stem cells are hematopoietic stem / progenitor cells (HSCs). In one embodiment, the stem cells are circulating blood cells. In one embodiment, the stem cells are mobilized blood cells. In one embodiment, the stem cells are bone marrow cells. In one embodiment, the stem cells are bone marrow progenitor cells. In one embodiment, the stem cells are lymphoid progenitor cells. In one embodiment, the stem cells are lymphoid progenitor cells. In one embodiment, the stem cells are multipotent progenitor cells. In one embodiment, the stem cells are lineage-specific progenitor cells. In one embodiment, the stem cells are endothelial cells. In one embodiment, the stem cells are mesenchymal stromal cells. In one embodiment, the stem cells are non-cord blood derived CD34 +In one embodiment, the stem cells are umbilical cord endothelial cells or umbilical cord blood cells. In one embodiment, the stem cells are umbilical cord blood CD34 + It is a cell.

[0156] In one embodiment, the stem cells are cultured in the presence of a cell viability enhancer (e.g., aryl hydrocarbon receptor, innate immune response antagonist, or other cell viability enhancer described herein) for 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 days or less, or for 96, 72, 60, 48, 36, 24, 12, 6, 3, 2, or 1 hour or less. In one embodiment, the stem cells are cultured in the presence of a stem cell viability enhancer for more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, 72, 84, or 96 hours. In one embodiment, the stem cells are cultured in the presence of a cell viability enhancer for less than 120 hours.

[0157] Aryl hydrocarbon receptor (AhR) antagonists In one embodiment, stem cell viability enhancer is aryl hydrocarbon receptor (AhR) antagonist.Aryl hydrocarbon receptor (AhR) is a transcription factor that mediates the response to environmental toxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) and other polychlorinated biphenyls, and is involved in stem cell proliferation and differentiation (see, for example, Thurmond et al. (1999) Toxicol.Appl.Pharmacol.158:33-40; Singh et al. (2009) Biochem.Pharmacol.77,577-587; Esser (2012) Arch.Toxicol.86:1323-1329).So far, several AhR antagonists have been identified.

[0158] Exemplary AhR antagonists for use disclosed herein include, but are not limited to, Stem Regenin 1 (Boitano et al. 2010), dimethoxyflavone (Carlin et al. 2013), 6,2',4'-trimethoxyflavone (CAS No. 720675-74-1; Murray et al. (2010) J. Pharmacol. Exp. Ther. 332(1):135-44), CH223191 (also known as 1-methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide; CAS No. 301326-22-7), α-naphthoflavone (ANF or α-NF), LGC006 (Boitano et al. al. 2010); 1-amino-3,7,8-trichlorodibenzo-p-dioxin (Luster et al. (1986) Biochem Biophys Res Commun. 139:747-56), 3'methoxy-4'-nitroflavone (Henry et al. 1999) Mol. Pharmacol. 55:716-25), resveratrol (Ciolino et al. (1998) Cancer Res. 58:5707-12), and GNF-351 (also known as N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; see, e.g., Smith et al. (2011) J. Pharmacol. Exp. Ther. 338:318-27). Other AhR antagonists, including analogs of the AhR antagonists disclosed herein, will be known to those of skill in the art.

[0159] In one embodiment, the AhR antagonist is StemRegenin-1 (SR1), a small molecule (4-[2-[[2-benzo[b]thien-3-yl-9-(1-methylethyl)-9H-purin-6-yl]amino]ethyl]-phenol) that was identified in a screen for molecules that direct HSC expansion (Boitano et al. 2010, Science 329:1345-1348).

[0160] In certain embodiments, stem cells are contacted with one AhR antagonist. In certain embodiments, stem cells are contacted with two or more AhR antagonists. In certain embodiments, stem cells are contacted with one or more AhR antagonists before contacting the cells with CRISPR / Cas9 components. In certain embodiments, stem cells are contacted with one or more AhR antagonists before and after contacting the cells with CRISPR / Cas9 components. In some embodiments, stem cells are contacted with at least one first AhR antagonist before contacting the cells with CRISPR / Cas9 components, and further contacted with a second AhR antagonist after contacting the cells with CRISPR / Cas9 components. In some embodiments, the first and second AhR antagonists are different AhR antagonists.

[0161] The appropriate concentration of AhR antagonist for use in the methods disclosed herein will be readily apparent to those skilled in the art (see, e.g., Merchant et al. (1990) Arch. Biochem. Biophys. 281:84-9; and Gasiewicz et al. (1991) Mol. Pharmacol. 40:607-12). In some embodiments, stem cells are contacted with a medium containing less than 5, 4, 3, 2, or 1 μM of AhR antagonist. In some embodiments, stem cells are contacted with a medium containing less than 900, 800, 700, 600, 500, 400, 300, 200, 100, or 50 nM of AhR antagonist. In some embodiments, the stem cells are contacted with a medium comprising 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nM of an AhR antagonist. In some embodiments, the stem cells are contacted with a medium comprising at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, or 500 nM of an AhR antagonist.

[0162] In some embodiments, stem cells are contacted with a medium containing both an AhR antagonist and a cytokine (e.g., a cytokine disclosed herein). In some embodiments, stem cells are contacted with a medium containing both an AhR antagonist and a pyrimidoindole derivative (e.g., UM729, UM171). For example, in one embodiment, stem cells are contacted with SR1 and UM729. In another embodiment, stem cells are contacted with SR1 and UM171.

[0163] Innate immune response In another embodiment, the stem cell viability enhancer is an innate immune response antagonist. The term "innate immune response antagonist," as used herein, refers to a molecule that inhibits a stem cell's innate immune response (e.g., in response to contact of a stem cell with one or more components of a Cas9 system (e.g., by electroporation)). In some embodiments, the stem cell viability enhancer inhibits signaling events necessary for a stem cell's innate immune response (e.g., in response to contact of a stem cell with one or more components of a Cas9 system (e.g., by electroporation)) to occur. In some embodiments, the stem cell viability enhancer inhibits cell death (e.g., programmed cell death) of a stem cell. In some embodiments, the stem cell viability enhancer inhibits programmed cell death of a stem cell (e.g., in response to contact of a stem cell with one or more components of a Cas9 system). In some embodiments, the stem cell viability enhancer inhibits signaling events necessary for a cell to undergo programmed cell death signaling events (e.g., in response to contact of a stem cell with one or more components of a Cas9 system). In some embodiments, the stem cell viability enhancer inhibits senescence in stem cells. In some embodiments, the stem cell viability enhancer inhibits differentiation of stem cells. In one embodiment, the stem cell viability enhancer inhibits apoptosis of stem cells. In one embodiment, the stem cell viability enhancer inhibits autophagy of stem cells. In some embodiments, the stem cell viability enhancer increases the frequency of stem cell inoculation into a target tissue compared to the frequency of stem cell inoculation into the target tissue without treatment with the stem cell viability enhancer.

[0164] Examples of innate immune response antagonists are known in the art. Exemplary innate immune response antagonists for use disclosed herein include, but are not limited to, cyclosporin A, TLR-4C34 (3,4,6-triacetate-1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside; also known as CAS No. 40592-88-9), CLI-095 (also known as TAK-242 or resatorbid), the TLR-4 inhibitor peptide VIPER (see, e.g., Lysakova-Devine et al. (2010) J. Immunol. 2010; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022; 2023; 2024; 2025; 2026; 2027; 2028; 2030; 2031; 2032; 2033; 2034; 2035; 2036; 2037; 2038; 2039; 2040; 2041; 2042; 2043; 2044; 2045; 2046; 2047; 2048; 2049; 2050; 2051; 2052; 2053; 2054; 2055; 2056; 2057; 2058; 2060; 2061; 2062; 2063; 2064; 2065; 2066; 2067 Immunol. 185:4261-4271), resveratrol, nitric oxide, carbocysteine, CGS25462, CHS828, clarithromycin, dipyridamole, disulfiram, diltiazem, fenoldopam, fibrates, fluvastatin, Gleevec, leflunomide, moxifloxacin, perindopril, raloxifene, rapamycin, ritonavir, tetrathiomolybdate, triflusal, troglitazone, MyD88 inhibitor peptides, and R-MyD88 targeting peptides. NAi agents, B18R recombinant protein, glucocorticoids (e.g., dexamethasone), 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), TLR antagonists, rapamycin, bafilomycin, BX795, FK506 (tacrolimus), and retinoic acid-inducible gene 1-like receptor (RLR) inhibitors (e.g., small molecules that inhibit or reduce the expression of RLR receptors described herein, or inhibitory nucleic acids (i.e., shRNA or siRNA)). In one embodiment, the innate immune response antagonist is cyclosporin A. In another embodiment, the innate immune response antagonist is dexamethasone. In one embodiment, the innate immune response antagonist is resveratrol.In another embodiment, the innate immune response antagonist is the transforming growth factor-β (TGF-β) type I receptor activin receptor-like kinase ALK5 inhibitor SB431542 (also known as 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide; CAS No. 301836-41-9; see, e.g., Inman et al. Mol. Pharmacol. 62(1):65-74). In one embodiment, the innate immune response antagonist is the protease inhibitor MG132 (also known as benzyl (S)-4-methyl-1-((S)-4-methyl-1-((S)-4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2-ylamino)-1-oxopentan-2-ylcarbamate; see, e.g., Tsubuki et al. (1996) J. Biochem. 119:572-6). Without wishing to be bound by any theory, the use of proteasome inhibitors may be particularly advantageous in preventing degradation of Cas9 molecules to which stem cells are exposed. In another embodiment, the innate immune response antagonist is the MyD88 inhibitor peptide PepinH-MYD (NBP2-29328, Novus Biologicals). In another embodiment, the innate immune response antagonist is IMO-8400 (see, e.g., Suarez-Farinas et al. (2013) PLoS One 8(12):e84634). In one embodiment, the innate immune response antagonist is a nucleic acid inhibitor of MyD88 (e.g., shRNA, siRNA, antisense RNA that inhibits or reduces MyD88 expression). In one embodiment, the innate immune response antagonist is B18R protein (also known as vaccinia virus-encoded neutralizing type I interferon receptor or type I IFN inhibitor; see, e.g., Vancova et al. (1998) J. Gen. Virol. 79(Pt 7):1647-9).In one embodiment, the innate immune response antagonist is a glucocorticoid (e.g., aldosterone, deoxycorticosterone acetate, fludrocortisone acetate, beclomethasone, triamcinolone, betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, cortisone, and hydrocortisone). In some embodiments, the innate immune response antagonist is oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC). In one embodiment, the innate immune response antagonist is a nucleic acid inhibitor of a Toll-like receptor (TLR) (e.g., an shRNA, siRNA, or antisense RNA that inhibits or reduces the expression of a TLR described herein). In another embodiment, the innate immune response antagonist is rapamycin. In one embodiment, the innate immune response antagonist is the mTOR inhibitor bafilomycin. In another embodiment, the innate immune response antagonist is N-[3-[[5-indo-4-[[3-[(2-thienylcarbonyl)amino]propyl]amino]-2-pyrimidinyl]amino]phenyl]-1-pyrrolidinecarboxamide (BX795; CAS No. 702675-74-9). In one embodiment, the innate immune response antagonist is a retinoic acid-inducible gene I-like (RIG-I-like) receptor (RLR) inhibitor. In one embodiment, the RLR-I-like receptor is a receptor encoded by a gene selected from the group consisting of RIG-I, MDA-5, LGP2, STING, DAK, and RNF125. In one embodiment, the RLR-I-like receptor inhibitor is an inhibitor of the receptor encoded by the gene RIG-I (e.g., a small molecule that inhibits or reduces the expression of RIG-I, or an inhibitory nucleic acid (i.e., shRNA, siRNA)). In another embodiment, the RLR-I-like receptor inhibitor is an inhibitor of the receptor encoded by the gene MDA-5 (e.g., a small molecule or inhibitory nucleic acid (i.e., shRNA, siRNA) that inhibits or reduces expression of MDA-5). In another embodiment, the RIG-I-like receptor inhibitor is an inhibitor of the receptor encoded by the gene LGP2 (e.g., a small molecule or inhibitory nucleic acid (i.e., shRNA, siRNA) that inhibits or reduces expression of LGP2).In another embodiment, the RLR-I-like receptor inhibitor is an inhibitor of the receptor encoded by the gene STING (e.g., a small molecule or inhibitory nucleic acid (i.e., shRNA, siRNA) that inhibits or reduces the expression of STING). In another embodiment, the RIG-I-like receptor inhibitor is an inhibitor of the receptor encoded by the gene SAK (e.g., a small molecule or inhibitory nucleic acid (i.e., shRNA, siRNA) that inhibits or reduces the expression of DAK). In another embodiment, the RIG-I-like receptor inhibitor is an inhibitor of the receptor encoded by the gene RNF125 (e.g., a small molecule or inhibitory nucleic acid (i.e., shRNA, siRNA) that inhibits or reduces the expression of RNF125). In one embodiment, the innate immune response antagonist is GW788388 (4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide; also known as CAS No. 452342-67-5). In one embodiment, the innate immune response antagonist is (4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide; 6-quinolinecarboxamide, 4-[5,6-dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl], LY-2157299; also known as CAS No. 7008474-72-2). In one embodiment, the innate immune response antagonist is fresolumimab (also known as GC1008). In another embodiment, the innate immune response antagonist is TLR-4C34 (also known as 3,4,6-triacetate-1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside; CAS No. 40592-88-9). In one embodiment, the innate immune response antagonist is CLI-095 (also known as TAK-242 or resatorbid). In another embodiment, the innate immune response antagonist is the TLR-4 inhibitor peptide VIPER.

[0165] In one embodiment, the innate immune response antagonist is resveratrol. In another embodiment, the innate immune response antagonist is nitric oxide. In another embodiment, the innate immune response antagonist is carbocysteine. In another embodiment, the innate immune response antagonist is CGS 25462.

[0166] In one embodiment, the innate immune response antagonist is CHS 828. In another embodiment, the innate immune response antagonist is clarithromycin. In one embodiment, the innate immune response antagonist is dipyridamole. In another embodiment, the innate immune response antagonist is disulfiram. In one embodiment, the innate immune response antagonist is diltiazem. In another embodiment, the innate immune response antagonist is fenoldopam. In one embodiment, the innate immune response antagonist is a fibrate. In another embodiment, the innate immune response antagonist is fluvastatin. In one embodiment, the innate immune response antagonist is Gleevec. In another embodiment, the innate immune response antagonist is leflunomide. In one embodiment, the innate immune response antagonist is peridopril. In another embodiment, the innate immune response antagonist is moxifloxacin. In one embodiment, the innate immune response antagonist is raloxifene. In another embodiment, the innate immune response antagonist is rapamycin. In one embodiment, the innate immune response antagonist is ritonavir. In another embodiment, the innate immune response antagonist is tetrathiomolybdate. In one embodiment, the innate immune response antagonist is triflusal. In another embodiment, the innate immune response antagonist is troglitazone. In one embodiment, the innate immune response antagonist is lovastatin. In another embodiment, the innate immune response antagonist is NR-101 (Nitino et al. (2009) Exp. Hematol. 37(11):1364-77). In one embodiment, the innate immune response antagonist is mir-125a (see, e.g., Pan et al. (2015) Nat. Commun. 6:7096). In one embodiment, the innate immune response antagonist is an Ndr kinase modulator.

[0167] In one embodiment, the innate immune response antagonist is an NFκB inhibitor (e.g., an NFκB inhibitor disclosed in Table 1a). Without wishing to be bound by theory, an NFκB inhibitor can be used to maintain stem cell viability and / or reduce the innate immune response associated with exposure to CRISPR / Cas9 components. In one embodiment, the NFκB inhibitor is dexamethasone. In another embodiment, the NFκB inhibitor is reservatrol. In one embodiment, the NFκB inhibitor is SB431542. In another embodiment, the NFκB inhibitor is MG132. In another embodiment, the NFκB inhibitor is carbocysteine. In one embodiment, the NFκB inhibitor is CGS25462. In another embodiment, the NFκB inhibitor is CHS828. In one embodiment, the NFκB inhibitor is clarithromycin. In another embodiment, the NFκB inhibitor is dipyridamole. In one embodiment, the NFκB inhibitor is disulfiram. In another embodiment, the NFκB inhibitor is diltiazem. In one embodiment, the NFκB inhibitor is fenoldopam. In another embodiment, the NFκB inhibitor is a fibrate. In one embodiment, the NFκB inhibitor is fluvastatin. In another embodiment, the NFκB inhibitor is Gleevec. In one embodiment, the NFκB inhibitor is leflunomide. In another embodiment, the NFκB inhibitor is moxifloxacin. In one embodiment, the NFκB inhibitor is perindopril. In another embodiment, the NFκB inhibitor is raloxifene. In one embodiment, the NFκB inhibitor is rapamycin. In another embodiment, the NFκB inhibitor is ritonavir. In one embodiment, the NFκB inhibitor is tetrathiomolybdate. In another embodiment, the NFκB inhibitor is triflusal. In one embodiment, the NFκB inhibitor is troglitazone. In one embodiment, the NFκB inhibitor is denosumab.

[0168] Without wishing to be bound by theory, in one embodiment, it is believed that contacting stem cells with an immunomodulatory compound (e.g., an innate immune response antagonist) described herein can modulate stem cell self-renewal, maintenance, and survival, for example, by inhibiting the cellular innate immune response and subsequent cellular senescence response induced by gene editing associated with DNA damage. In another embodiment, contacting stem cells with an innate immune response antagonist described herein can inhibit activation of stem cell programmed cell death induced by contact of the cell with CRISPR / Cas9 components.

[0169] For example, inflammatory cytokines (e.g., interferon [IFN]), tumor necrosis factor alpha (TNFα), and toll-like receptors (TLRs) can directly affect stem cells, such as HSCs, allowing them to support effective immune responses while maintaining hematopoiesis in the peripheral blood. For example, inflammatory cytokines can activate HSCs in vivo to participate in the immune response by directing differentiation toward myeloid or lymphoid progeny based on cellular events necessary to delay invading pathogens (e.g., increased production of myeloid neutrophils as innate immune antimicrobial effector cells). Therefore, programmed cell death (e.g., apoptosis) can be prevented by contacting stem cells with an innate immune response antagonist. Exemplary innate immune response components and innate immune response antagonists are listed in Table 1a. Additional innate immune response antagonists are listed in Table 1b.

[0170] [Table 1]

[0171] [Table 2]

[0172] [Table 3]

[0173] [Table 4]

[0174] Additionally, certain stem cells, e.g., HSCs, express TLR receptors such as 4, 7, 8, and 9 (reviewed in Baldridge et al. (2011) Trends Immunol. 32(2):57-65). TLR7 / 8 and TLR3 sense double-stranded and single-stranded ribonucleic acid, respectively, and TLR9 sense deoxyribonucleic acid. Retinoic acid-inducible gene-like receptors (e.g., RIG-I) are RNA helicases that sense RNA and can induce an interferon response (reviewed in Kajaste-Rudnitski and Naldini (2015) Human Gene Therapy 26:201-209). TLR signaling can trigger inflammatory responses via NFκB signaling. Therefore, by blocking these factors involved in the innate immune response in stem cells, e.g., HSCs (e.g., by blocking interferon or transcription factors such as IRF3 to prevent an interferon response), programmed cell death (e.g., apoptosis) of stem cells induced by contact with, for example, the CRISPR / Cas9 components described herein can be prevented. Clinically approved immunomodulatory compounds cyclosporine A (CsA) and FK506 (Tacrolimus), both of which inhibit Ca 2+Inhibition of calcineurin activation prevents the expression of inflammatory cytokines. Rapamycin, a standard inducer of autophagy through inhibition of the mammalian target of rapamycin (mTOR) complex, has been shown to improve lentiviral transduction of HSCs without inducing autophagy or impairing cell engraftment (Wang et al. (2014) Blood 24:9130-923).

[0175] In one embodiment, the stem cells are contacted with an innate immune response inhibitor. In one embodiment, the immunomodulatory compound is a compound that regulates, for example, inhibits, a pathway or target listed in Table 1a or Table 1b. The stem cells may be contacted with the immunomodulatory compound before, during, and / or after contact with CRISPR / Cas9 components, for example, Cas9 molecules, gRNA molecules, or both, and optionally with a donor template nucleic acid.

[0176] In certain embodiments, the stem cells are contacted with one innate immune response antagonist. In certain embodiments, the stem cells are contacted with two or more innate immune response antagonists. In certain embodiments, the stem cells are contacted with one or more innate immune response antagonists before contacting the cells with CRISPR / Cas9 components. In one embodiment, the stem cells are contacted with one or more innate immune response antagonists after contacting the cells with CRISPR / Cas9 components. In certain embodiments, the stem cells are contacted with one or more innate immune response antagonists before and after contacting the cells with CRISPR / Cas9 components.

[0177] In some embodiments, the stem cells are contacted with at least one first innate immune response antagonist before contacting the cells with the CRISPR / Cas9 components, and the stem cells are further contacted with a second innate immune response antagonist after contacting the cells with the CRISPR / Cas9 components. In some embodiments, the first and second innate immune response antagonists are different innate immune response antagonists. In other embodiments, the first and second innate immune response antagonists are the same innate immune response antagonist.

[0178] Other stem cell viability enhancers In certain embodiments, the stem cell viability enhancer is selected from the group consisting of UM171, UM729, and 16,16-dimethylprostaglandin E2. In one embodiment, the stem cell viability enhancer is UM171 ((1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine; CAS No. 1448724-09-1; see, e.g., Fares et al. (2014) Science 345(6203):1509-1512). In another embodiment, the stem cell viability enhancer is UM729 (methyl 4-((3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate; CAS No. 1448723-60-1; see, e.g., Fares et al. (2014) Science 345(6203):1509-1512). In yet another embodiment, the stem cell viability enhancer is 16,16-dimethyl prostaglandin E2 (9-oxo-11α,15R-dihydroxy-16,16-dimethyl-prosta-5Z,13E-dien-1-oic acid; dmPGE2; CAS No. 39746-25-3). In one embodiment, the stem cell viability enhancer is prostaglandin E2 (PGE2). In another embodiment, the stem cell viability enhancer is MG132. In yet another embodiment, the stem cell viability enhancer is SB431542.

[0179] cytokines After the stem cells are obtained, generated, and / or isolated, but before contacting the cells with the CRISPR / Cas9 components (e.g., the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template nucleic acid), the stem cells can be cultured in a medium containing, for example, one or more cytokines in addition to a stem cell viability enhancer. Further, after contacting the cells with the CRISPR / Cas9 components, the stem cells can be cultured in a medium containing, for example, one or more cytokines in addition to a stem cell viability enhancer.

[0180] Without wishing to be bound by theory, culturing cells in medium further comprising the cytokines disclosed herein may induce cell cycle entry of the cells. Exemplary cytokines include, but are not limited to, stem cell factor (SCF), thrombopoietin (TPO), or human Flt-3 ligand (FL), interleukin-6 (IL-6), interleukin-11 (IL-11), insulin-like growth factor binding protein 1 (IGFBP1); insulin-like growth factor binding protein 2 (IGFBP2), angiopoietin-like protein 1 (ANGPTL1), angiopoietin-like protein 3 (ANGPTL3), angiopoietin-like protein 4 (ANGPTL4), and angiopoietin-like protein 5 (ANGPTL5) (see, e.g., Fan et al. (2014) Stem Cell Res. Ther. 5:71-80; and Blank et al. (2012) Eur. J. Haematol. 89:198-205).

[0181] In one embodiment, the stem cells are cultured in a medium containing one or more cytokines. In one embodiment, the stem cells are cultured in a medium containing two or more cytokines. In one embodiment, the stem cells are cultured in a medium containing three or more cytokines. In one embodiment, the stem cells are cultured in a medium containing four or more cytokines. In one embodiment, the stem cells are cultured in a medium containing five or more cytokines.

[0182] In certain embodiments, stem cells are cultured in a medium containing one or more cytokines before contacting the cells with CRISPR / Cas9 components. In certain embodiments, stem cells are cultured in a medium containing one or more cytokines after contacting the cells with CRISPR / Cas9 components. In certain embodiments, stem cells are cultured in a medium containing one or more cytokines before and after contacting the cells with CRISPR / Cas9 components. In one embodiment, stem cells are cultured in a medium containing cytokines (e.g., cytokines described herein) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In one embodiment, stem cells are cultured in a medium containing cytokines (e.g., cytokines described herein) for less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In one embodiment, stem cells are cultured for 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more than 14 days in a medium containing a cytokine (e.g., a cytokine described herein). For example, stem cells (e.g., stem or progenitor cells, e.g., hematopoietic stem / progenitor cells) can be cultured for 1-3 days in the following medium: StemSpan SFEM (Stem Cell Technologies) containing 1% penicillin / streptomycin and supplemented with 50-100 ng / mL each of human stem cell factor (SCF), human thrombopoietin (TPO), and human Flt-3 ligand (FL). The medium may further include one or more of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or interleukin-11 (IL-11).

[0183] Signal transduction activators or repressors After the stem cells are obtained, generated, and / or isolated, but before contacting the cells with the CRISPR / Cas9 components (e.g., the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template nucleic acid), the stem cells can be cultured in a medium containing, for example, one or more signaling activators or repressors in addition to the stem cell viability enhancer. Further, after contacting the cells with the CRISPR / Cas9 components, the stem cells can be cultured in a medium containing, for example, one or more signaling activators or repressors in addition to the stem cell viability enhancer.

[0184] In one embodiment, the stem cells are further contacted with a signaling activator or repressor before contact with the CRISPR / Cas9 components. In another embodiment, the stem cells are further contacted with a signaling activator or repressor after contact with the CRISPR / Cas9 components. In another embodiment, the stem cells are further contacted with a signaling activator or repressor both before and after contact with the CRISPR / Cas9 components.

[0185] The following signaling pathways are involved in the balance of stem cell (e.g., HSC) production, self-renewal, maintenance, proliferation, and cell fate determination. Important pathways regulating stem cells (e.g., HSC) include the Notch, TGFβ-SMAD, CXCR5, and Wnt signaling pathways (Mendelsen and Frenette (2014) Nature Medicine 20(8):833-46). Certain stem cells (e.g., HSC) express Notch, Wnt, and TGFβ receptors on the cell surface, and the self-renewal, proliferation, and differentiation capabilities of these cells can be altered by the binding of cognate ligands to these receptors on stem cells. Coordinated sequential or simultaneous regulation (e.g., activation or suppression) of these signaling pathways in stem cells (e.g., HSC) can be used to maintain the self-renewal, multipotency, and proliferation capabilities that can be perturbed upon contact of stem cells with CRISPR / Cas9 components or a delivery system for CRISPR / Cas9 components. Without wishing to be bound by theory, in one embodiment, it is believed that brief pre-treatment (e.g., before contact with CRISPR / Cas9 components) and post-treatment (e.g., after contact with CRISPR / Cas9 components) of stem cells with signaling pathway activators or inhibitors can promote cell survival and prevent cell death. In one embodiment, the methods described herein use signaling ligands that bind to cognate receptors on stem cells to maintain stem cell multipotency, self-renewal, and proliferation, while preventing senescence and cell death (e.g., apoptosis, autophagy, or necrosis). Table 2 lists exemplary signaling pathways and modulators thereof that can be used in accordance with the methods described herein, each of which is described in further detail in the following subsections.

[0186] [Table 5]

[0187] a. Notch signaling pathway Vascular endothelial cells and stromal cells in the bone marrow microenvironment produce membrane-bound Notch ligands (Delta-like ligand [DLL] and Jagged [JAG]) that bind to cognate receptors on specific stem cells, such as HSCs. Notch ligands compete for binding with Notch1 and Notch2 receptors on stem cells (e.g., HSCs), and the relative binding of various Notch ligands balances stem cell (e.g., HSC) self-renewal, maintenance, proliferation, and differentiation. For example, JAG1 has been shown to be required for homeostatic and regenerative hematopoiesis in vivo (Poulos et al. (2013) Cell Reports 4(5):1022-1034). Immobilized Notch ligand Delta-like ligand 1 (DLL1) (Immobilized Delta-1) on plastic culture plates was used to express the Notch ligand Delta-like ligand 1 (DLL1). ext-IgG ) and cord blood HSC co-cultures were performed using CD34 + It supports cell expansion and has been proven safe in an allogeneic double cord blood transplant clinical trial (Delaney et al. (2010) Nature Medicine 16:232-6).

[0188] In one embodiment, stem cells are contacted with one or more Notch signaling modulators. In one embodiment, the Notch signaling modulator is a Notch ligand, e.g., DLL or JAG. In one embodiment, the Notch signaling modulator is an eiCas9 molecule fused to an RNAi agent, an oligonucleotide, an antibody, or an activator / repressor in combination with a Notch pathway-specific gRNA. Stem cells are contacted with a Notch signaling modulator before, during, and / or after contact with CRISPR / Cas9 components, e.g., a Cas9 molecule, a gRNA molecule, or both, and, optionally, a donor template nucleic acid. Without wishing to be bound by theory, in one embodiment, contacting stem cells with a Notch signaling modulator is believed to regulate stem cell entry or exit into or out of the cell cycle to maintain cell viability and support limited stem cell proliferation. The methods described herein enable long-term engraftment of stem cells into human patients.

[0189] b TGFβ signaling pathway Transforming growth factor-β (TGFβ) is a growth factor present in the hematopoietic stem / progenitor cell niche that regulates HSC quiescence and hibernation (Yamzaki, 2009, Blood, 113:1250-1255). TGFβ is produced by nonmyelinating Schwann cells near blood vessels in the bone marrow (Yamazaki et al., 2011, Cell, 147:1146-1158), indicating that regulation of HSC quiescence occurs within the vascular (endothelial) niche. Treatment of zebrafish embryos with a small molecule inhibitor of TGFβ signaling (SB431542) has been shown to promote HSC proliferation within the perivascular niche (Tamplin et al., 2014, Cell, 160:241-252). Therefore, blocking TGFβ signaling with neutralizing antibodies or small molecule inhibitors, such as SB431542, is a strategy for maintaining the proliferative capacity of stem cells, such as HSCs, upon exposure to exogenous nucleic acids, such as Cas9 mRNA or gRNA. Alternatively, to prevent cell death due to brief exposure to exogenous nucleic acids, contact of stem cells (e.g., HSCs) with TGFβ immediately after exposure to genotoxic stress can force stressed stem cells into hibernation.

[0190] In one embodiment, stem cells are contacted with one or more TGFβ signaling modulators. In one embodiment, the TGFβ signaling modulator is a TGFβ ligand. In another embodiment, the TGFβ signaling modulator is a TGFβ signaling antagonist, e.g., SB43152 or an anti-TGFβR antibody. The stem cells are contacted with the TGFβ signaling modulator before, during, and / or after contact with CRISPR / Cas9 components, e.g., a Cas9 molecule, a gRNA molecule, or both, and, optionally, a donor template nucleic acid. In one embodiment, the TGFβ signaling modulator is an eiCas9 molecule fused to an RNAi agent, oligonucleotide, antibody, or activator / repressor in combination with a TGFβ pathway-specific gRNA. Without wishing to be bound by theory, in one embodiment, contacting stem cells with a TGFβ signaling modulator is believed to regulate stem cell entry or exit into or out of the cell cycle to maintain cell viability and support limited stem cell proliferation.

[0191] c.CXCR4 chemokine receptor CXCR4 is a chemokine receptor present on the cell surface of lymphocytes and HSCs. Stromal-derived factor-1 (SDF1 or CXCL12) is a ligand that binds to CXCR4 and is produced by cells in the bone marrow and other tissues. As cells migrate downward along a chemokine gradient, the SDF1 / CXCR4 signaling axis regulates HSC localization and retention in the bone marrow and migration into peripheral blood and other tissues. Small molecules have been developed to regulate HSC migration by utilizing the SDF1 / CXR4 chemokine axis. For example, the use of dmPGE2 upregulates the expression of CXCR4, enhancing the homing and retention of umbilical cord blood cells to the bone marrow during transplantation (Cutler et al., 2013, Blood, 122(17):3074-3081). As another example, plerixiflor (AMD3100), a CXCR4 antagonist that disrupts the binding of CXCR4 to SDF1, promotes mobilization of HSCs from bone marrow to blood for collection and use in allogeneic or autologous transplantation. Given that CXCR4 signaling plays a role in cell cycle progression and survival, manipulation of CXCR4 signaling in stem cells, e.g., ex vivo, can be used to prevent stem cell death (e.g., apoptosis) as well as regulate cell cycle progression and proliferation.

[0192] In one embodiment, stem cells are contacted with a CXCR4 modulator. In one embodiment, the CXCR4 modulator is a CXCR4 agonist, for example, dmPGE2. In another embodiment, the CXCR4 modulator is a CXCR4 antagonist, for example, plerixiflor (AMD3100). Stem cells are contacted with a CXCR4 modulator before, during, and / or after contact with a CRISPR / Cas9 component, for example, a Cas9 molecule, a gRNA molecule, or both, and optionally with a donor template nucleic acid. Without wishing to be bound by theory, in one embodiment, it is believed that contacting stem cells with a CXCR4 modulator can maintain the self-renewal, multipotency, or proliferation ability of stem cells.

[0193] d.Wnt signaling pathway Overexpression of the canonical Wnt pathway inhibitor DKK1 can reduce hematopoietic reconstitution in vivo (Fleming et al., 2008, Cell Stem Cell, 2(3):274-283). Deletion of the canonical Wnt ligand Wnt3a can also reduce the self-renewal and reconstitution capacity of HSCs. These findings indicate a specific role for canonical Wnt signaling in HSC self-renewal and maintenance. Conversely, non-canonical Wnt signaling (e.g., Wnt5a ligand) can inhibit canonical Wnt signaling in HSCs, blocking their ex vivo proliferation and increasing their repopulation capacity (Nemeth et al., 2007, Proceedings of the National Academy of Sciences, 104(39):15436-41).

[0194] Certain stem cells, such as HSCs, express non-canonical Wnt receptors. Binding of Wnt ligands to non-canonical Wnt receptors (e.g., Frizzled 8 [Fzd8]) prevents the nuclear localization of nuclear factor of activated T cells (NFAT), thereby preventing interferon expression and maintaining HSC quiescence (Sugimura et al., 2012, Cell, 150(2):351-365). Stress associated with viruses, foreign nucleic acids or proteins, perturbations in oxygen tension (e.g., culturing cells under hyperoxic or hypoxic conditions), or exposure of cells to electric fields (e.g., electroporation of cells to deliver foreign nucleic acids or proteins) can alter the balance between non-canonical Wnt signaling (which prevents cell activation and maintains self-renewal) and canonical Wnt signaling (which activates cells and induces cell proliferation, which can lead to exhaustion). Certain stem cells, such as HSCs, can be highly sensitive to Wnt signaling.

[0195] Thus, careful titration of Wnt ligands can be used to maintain specific stem cells, e.g., HSCs, while preventing stress responses that can occur upon contact with CRISPR / Cas9 components delivered, e.g., by electroporation or virus.

[0196] In one embodiment, stem cells are contacted with one or more modulators of Wnt signaling. In one embodiment, stem cells are contacted with a canonical Wnt ligand. In another embodiment, stem cells are contacted with a non-canonical Wnt ligand. In yet another embodiment, stem cells are contacted with an inhibitor of the Wnt signaling pathway. Exemplary inhibitors of the Wnt signaling pathway include, but are not limited to, neutralizing antibodies against specific Wnt receptors, or RNAi agents (e.g., shRNAs) against downstream targets of Wnt signaling, including, but not limited to, NFAT and IFNγ. Stem cells are contacted with a Wnt signaling modulator before, during, and / or after contact with CRISPR / Cas9 components, such as a Cas9 molecule, a gRNA molecule, or both, and optionally a donor template nucleic acid.

[0197] Inhibitors of programmed cell death After the stem cells are obtained, generated, and / or isolated, but before contacting the cells with the CRISPR / Cas9 components (e.g., the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template nucleic acid), the stem cells can be cultured in a medium containing, for example, one or more inhibitors of cell death in addition to the stem cell viability enhancer. Further, after contacting the cells with the CRISPR / Cas9 components, the stem cells can be cultured in a medium containing, for example, one or more inhibitors of cell death in addition to the stem cell viability enhancer.

[0198] In one embodiment, the stem cells are further contacted with a molecule that inhibits programmed cell death or senescence, for example, in a medium, before contacting with the molecule. In one embodiment, the stem cells are contacted with the compound within 24 hours (e.g., within 12 hours or 6 hours) before contacting with the CRISPR / Cas9 components, such as the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template molecule. In another embodiment, the stem cells are contacted with the compound within 24 hours (e.g., within 12 hours or 6 hours) after contacting with the CRISPR / Cas9 components, such as the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template molecule. In another embodiment, the stem cells are contacted with the compound within 24 hours (e.g., within 12 hours or 6 hours) before and within 24 hours (e.g., within 12 hours or 6 hours) after contacting with the CRISPR / Cas9 components, such as the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template molecule. In one embodiment, brief pre-treatment (e.g., before contact with CRISPR / Cas9 components) and post-treatment (e.g., after contact with CRISPR / Cas9 components) of stem cells with compounds that inhibit programmed cell death or senescence can increase the survival or proliferative capacity of stem cells upon exposure to exogenous nucleic acids, e.g., by temporarily inhibiting programmed cell death, including autophagy, apoptosis, and senescence.

[0199] In one embodiment, the stem cells comprise or are contacted with an eiCas9 molecule fused to, for example, a transcriptional repressor or activator (e.g., KRAB) to regulate a target gene associated with programmed cell death or senescence. Without wishing to be bound by theory, in one embodiment, for example, regulation of a target gene associated with programmed cell death or senescence by an eiCas9 molecule can enhance cell lifespan, viability, and proliferation without loss of multipotency, while maintaining cytocompatibility upon brief exposure to CRISPR / Cas9 components.

[0200] In one embodiment, stem cells are contacted with a combination of stem cell viability enhancers together with cytokines and / or signal transduction activators or repressors up to 120 hours (e.g., up to 96, 72, 60, 48, 36, or 24 hours) prior to contact with CRISPR / Cas9 components and up to 72 hours after contact with CRISPR / Cas9 components, e.g., to enhance gene editing and survival, reduce cell death (e.g., apoptosis, autophagy, or necrosis), or enhance engraftment. In one embodiment, rather than directly contacting with one of the cytokines or small molecules disclosed herein, stem cells are contacted with an inhibitor (e.g., an antibody) that binds to the cognate receptor of the cytokine or small molecule, e.g., a receptor listed in Table 3.

[0201] [Table 6]

[0202] Introduction of truncated cell surface antigens Purification of modified stem cells that express a cell surface antigen or selectable marker can provide a means of ensuring that the CRISPR / Cas9 components, e.g., the Cas9 molecule, the gRNA molecule, or both, and optionally the donor template nucleic acid, have been delivered to the cells, e.g., ex vivo. Expression of the cell surface antigen by the targeted cells also allows for tracking of the modified stem cells in vivo.

[0203] In one embodiment, the stem cells contain or are contacted with a gene encoding a cell surface antigen or selection marker. In one embodiment, the cell surface antigen or selection marker is truncated CD19 (tCD19). In another embodiment, the cell surface antigen or selection marker is truncated CD20 (tCD20). The full-length cell surface receptors CD19 and CD20 are naturally expressed on B lymphocytes. Cleavage of CD19 or CD20 prevents intracellular signaling through the receptor because the cytoplasmic domain is removed (Tey et al., 2007, Biol Blood Marrow Transplant, 13(8):913-24). Expression of the extracellular domain of CD19 or CD20 can enable cell sorting and in vivo cell tracking (e.g., by drawing blood and staining the cells with anti-human CD19 or anti-human CD20 antibodies to monitor the engraftment of gene-edited cells). In one embodiment, the tCD19 or tCD20 transgene is delivered as a donor template nucleic acid. In one embodiment, stem cells are contacted with one or more gRNA molecules comprising a targeting domain complementary to a targeting domain from the region where the transgene will be integrated. In one embodiment, a tCD19 or tCD20 transgene is integrated into the genome, for example, into a safe harbor locus, such as the AAVS1 safe harbor locus. Without wishing to be bound by theory, in one embodiment, the introduction or co-introduction of truncated CD19 or CD20 cell surface antigens (multiplex genome editing) may be used to purify genome-edited cells in vitro or monitor genome-edited cells in vivo.

[0204] Introduction of chemotherapy resistance transgenes or suicide genes The methods described herein allow for the modulation of stem cells in vivo or in vitro so that modified stem cells with desired characteristics can be selected or expanded, or modified stem cells with unwanted characteristics (e.g., leukemic transformation) can be eliminated.

[0205] In one embodiment, the stem cells comprise or are contacted with a safety switch that allows for the selection of desired stem cells, e.g., in vitro or in vivo, or the elimination of unwanted stem cells, e.g., in vitro or in vivo. In one embodiment, the safety switch contains a suicide gene and / or a gene encoding a chemotherapy selectable marker. For example, stem cells can contain a safety switch that includes two components: 1) a truncated cell surface antigen (tCD20) and an inducible suicide gene, which can be used to select genome-edited cells in vitro, track them in vivo, and eliminate cells in vivo if leukemic transformation occurs upon patient administration of rituximab (an anti-CD20 monoclonal antibody therapy); and 2) a drug-inducible chemotherapy resistance gene (e.g., a P140K mutant form of methylguanine methyltransferase [P140K MGMT]), which selects genome-edited cells in vivo by eliminating non-edited cells upon treatment of patients with alkylating chemotherapy drugs (O6-benzylguanine [O6BG] and BCNU), thereby enhancing in vivo repopulation of bone marrow with genome-edited cells.

[0206] In one embodiment, the stem cells comprise or are contacted with a suicide gene. In one embodiment, the suicide gene encodes inducible caspase-9 (iCasp9). In one embodiment, the stem cells are further contacted with a chemical inducer of dimerization, e.g., AP1903 or AP2018. Without wishing to be bound by theory, it is believed that caspase-9 induces apoptosis upon treatment with a chemical inducer of dimerization (Di Stasi et al., 2011, New Eng Journal Med, 365:1673-1683). In another embodiment, the suicide gene encodes truncated CD20 (tCD20). In one embodiment, the stem cells are further contacted with an anti-CD20 antibody, e.g., rituximab. Without wishing to be bound by theory, it is believed that anti-CD20 antibodies can induce an immune response that causes the death of cells that express CD20 (Redman et al., 2015, Mol Immunol, S0161-5890(15):00361-2).

[0207] In one embodiment, the stem cells comprise or are contacted with a gene encoding a chemotherapy selection marker. In one embodiment, the chemotherapy selection marker is a mutant form of methylguanine methyltransferase (e.g., a P140K mutant form of methylguanine methyltransferase). In one embodiment, the stem cells are further contacted with a chemotherapy agent, for example, O6BG / BCNU. Without wishing to be bound by theory, in one embodiment, the use of a P140K mutant form of methylguanine methyltransferase in combination with O6BG / BCNU chemotherapy is believed to be effective in increasing the level of gene-modified hematopoietic stem / progenitor cells in the bone marrow after delivery by lentiviral transduction (Gori et al., 2012, Cancer Gene Therapy, 19(8):1523-9; Beard et al., 2010. J Clin Invest, 120(7):2345-54).

[0208] In one embodiment, the transgene is provided on or delivered as a donor template nucleic acid. In one embodiment, the stem cell is contacted with one or more gRNA molecules comprising a targeting domain complementary to a targeting domain from the region where the transgene will be integrated. In one embodiment, the transgene is integrated into the genome, for example, into a safe harbor locus, such as the AAVS1 safe harbor locus. In one embodiment, the transgene comprises a tCD20-2A-P140K2 cistronic transgene cassette.

[0209] Stem cell culture As described herein, stem cells can be cultured either before, after, or both of contact with CRISPR / Cas9 components, e.g., Cas9 molecules, gRNA molecules, or both, and optionally with a donor template nucleic acid. In one embodiment, stem cells are co-cultured with endothelial cells, e.g., human endothelial cells (e.g., VeraVec™, perivascular endothelial, or other endothelial cells). In another embodiment, stem cells are co-cultured with mesenchymal stromal cells, e.g., human mesenchymal stromal cells. In one embodiment, the endothelial or mesenchymal co-cultured cells are genetically modified to express one or more membrane-bound ligands that activate or suppress Notch, Wnt, TGFβ, and / or CXCR4 signaling in the stem cells. In one embodiment, stem cells are cultured in a 2-D culture system. In another embodiment, stem cells are cultured in a 3-D culture system. For example, stem cells can be cultured in a system designed for culturing stem or progenitor cells, e.g., HSCs (e.g., NANEX™ expansion plates, AK-polyfiber). In one embodiment, stem cells are cultured under hypoxic growth conditions, e.g., by placing the cell culture vessel in a hypoxic chamber. For example, the oxygen tension in the hypoxic chamber can be in the range of 10% to 0.1% O2, e.g., 5% to 0.5% O2, e.g., 10% or less, 8% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0.2% or less O2. In one embodiment, stem cells are cultured for at least 1, 2, 3, 4, 5, 6, or 7 days. In one embodiment, stem cells are purified from endothelial or stromal support cells prior to transplantation.

[0210] Modification of gRNA and template molecules During virus-host coevolution, viral RNA capping, which mimics mRNA capping, evolved, allowing viral RNA to escape detection by the cell's innate immune system (see, e.g., Delcroy et al. (2012) Nature Reviews Microbiology 10:51-65). Toll-like receptors in stem cells (e.g., HSCs) detect the presence of foreign single- and double-stranded RNA, which can trigger innate immune responses, cellular senescence, and programmed cell death (Kajaste-Rudnitski and Naldini (2015) Human Gene Therapy 26:201-9). Initial experimental results demonstrated that human HSCs electroporated with unmodified gRNA molecules (e.g., gRNA synthesized without a 5' cap or 3' polyA-tail) and Cas9 mRNA exhibited reduced cell lifespan, proliferation, or multipotency (e.g., loss of erythroid differentiation potential and skewed myeloid differentiation potential) compared with cells electroporated with GFP mRNA alone. Cell senescence and apoptosis may be due to stem cell sensing of foreign nucleic acids and the induction of an innate immune response, followed by the induction of programmed cell death and the loss of proliferation and differentiation potential. To avoid the innate immune response of cells to foreign nucleic acids, gRNA molecules can be modified to mimic mRNA (e.g., by adding a 5' cap and a 3' polyA tail), thereby preventing the innate immune response in cells, the interferon response in cells, cellular senescence, or programmed cell death associated with sensing foreign nucleic acids.

[0211] "Modified gRNA molecule" or "modified gRNA," as used herein, refers to a gRNA molecule that has an improved half-life after being introduced into a cell compared to an unmodified gRNA molecule after being introduced into a cell. In one embodiment, the modified gRNA molecule does not activate an innate immune response in a cell when the cell is exposed to the gRNA molecule (e.g., electroporated). In one embodiment, the modified gRNA molecule activates a reduced innate immune response in a cell when the cell is exposed to the gRNA molecule compared to the innate immune response in the same cell when the cell is exposed to the unmodified gRNA. In another embodiment, the modified gRNA molecule does not activate a programmed cell death pathway (e.g., an apoptotic cell death pathway, a necrotic cell death pathway (e.g., a necrotic cell death pathway), an autophagy cell death pathway, an aponecrotic cell death pathway, a ferroptosis cell death pathway, an eryptosis cell death pathway, an aponecrotic cell death pathway, or an anoikis cell death pathway) in a cell when the cell is exposed to the gRNA. In some embodiments, the modified gRNA molecule does not activate a caspase-dependent cell death pathway. In other embodiments, the modified gRNA molecule does not activate a caspase-independent cell death pathway.

[0212] In one embodiment, the modified gRNA molecule comprises a 5'-end modification. In one embodiment, the 5'-end modification is selected from the group consisting of: G(5')ppp(5')G cap analog, m7G(5')ppp(5')G cap analog, or 3'-O-Me-m7G(5')ppp(5')G anti-reverse cap analog (ARCA). In one embodiment, the 5'-end modification is a phosphorothioate modification. In one embodiment, the gRNA molecule comprises a 3'-end modification. In one embodiment, the 3'-end modification is a polyadenine tail. In one embodiment, the 3'-end modification is a phosphorothioate modification.

[0213] In one embodiment, stem cells are contacted with a capped and tailed gRNA molecule. In one embodiment, the term "capped," as used herein to refer to a gRNA molecule, refers to a gRNA having a 5'-terminal cap structure. In some embodiments, the 5-terminal cap structure is selected from the group consisting of: G(5')ppp(5')G cap analog, mG(5')ppp(5')G cap analog, or 3'-O-Me-mG(5')ppp(5')G anti-reverse cap analog (ARCA). In one embodiment, the term "tailed," as used herein to refer to a gRNA molecule, refers to a gRNA having a 3'-terminal modification. In one embodiment, the 3'-terminal modification is a polyadenine tail. In one embodiment, the 3'-terminal modification is a phosphorothioate modification. In one embodiment, the gRNA molecule comprises a 3' poly-adenine tail. In certain embodiments, the poly-adenine tail is 5, 10, 15, 20, 25, 30, 35, 40 adenine residues in length or more. In one embodiment, stem cells are contacted with a Cas9 molecule / gRNA molecule complex containing a capped and tailed gRNA molecule. Without wishing to be bound by theory, in one embodiment, it is believed that contacting stem cells with a capped and tailed gRNA molecule can increase survival of the modified stem cells, maintain stem cell multipotency or viability, increase cell engraftment, or inhibit cell senescence and programmed cell death.

[0214] In one embodiment, the stem cell is further contacted with a donor template nucleic acid. In one embodiment, the donor template nucleic acid is a single-stranded oligodeoxynucleotide. Similar to the modified gRNA molecules described above, the donor template nucleic acid can be modified to include a 5'-end modification, a 3'-end modification, or both a 5'-end and a 3'-end modification to circumvent the cellular innate immune response to the foreign nucleic acid, thereby preventing the cellular innate immune response, the cellular interferon response, cellular senescence, or programmed cell death associated with the sensing of the foreign nucleic acid. In some embodiments, the donor template nucleic acid includes a 5'-phosphorothioate modification. In some embodiments, the donor template nucleic acid includes a 3'-phosphorothioate modification. In some embodiments, the donor template nucleic acid includes both a 5'-phosphorothioate modification and a 3'-phosphorothioate modification.

[0215] Methods for treating or preventing diseases The methods and compositions described herein provide therapies, e.g., single-dose or multi-dose therapies, for treating or preventing diseases, e.g., diseases described herein. In some embodiments, the method of treating or preventing a disease involves modifying cells, e.g., cells described herein, e.g., ex vivo or in vivo. Any type of cell associated with a disease can be modified using the methods described herein. For example, the cell is a circulating blood cell, a mobilized blood cell, a bone marrow cell, a myeloid progenitor cell, a lymphoid progenitor cell, a hematopoietic stem / progenitor cell (HSC), a multipotent progenitor cell, a lineage-specific progenitor cell, an endothelial cell, or a mesenchymal stromal cell. In another embodiment, the method of treating or preventing a disease involves modifying a gene, e.g., a gene described herein, e.g., by CRISPR / Cas-mediated gene editing. Cell or gene modification (e.g., correction, knockout, knock-in, knockdown, or activation) can be performed before or after the onset of the disease. Exemplary diseases that can be treated or prevented using the methods described herein include, but are not limited to, those listed in Table 4. Exemplary genes that can be modified by the methods described herein include, but are not limited to, the genes listed in Table 4.

[0216] In one embodiment, a gene is knocked into a safe harbor locus in a stem cell (e.g., the AAVS1 safe harbor locus), e.g., HSC, using CRISPR / Cas-mediated methods or any other knock-in or gene delivery method, including Sleeping Beauty transposon, lentiviral vectors, or adeno-associated viral vectors.

[0217] In one embodiment, the gene encodes a secreted soluble protein. Without wishing to be bound by theory, in one embodiment, it is believed that knock-in of a gene encoding a secreted soluble blood protein can be used to treat or cure diseases, including those listed in Table 4, such as lysosomal storage diseases, glycogen storage diseases, mucopolysaccharidoses, or any disease in which secretion of a protein ameliorates the disease. In one embodiment, the disease is sickle cell disease (SCD). In another embodiment, the disease is beta-thalassemia.

[0218] In one embodiment, the disease is associated with a deficiency of a circulating blood protein. Exemplary diseases include, but are not limited to, hemophilia (e.g., hemophilia A or hemophilia B), A1AT deficiency, or lysosomal acid lipase deficiency. Without wishing to be bound by theory, in one embodiment, it is believed that introducing a gene encoding a secreted soluble blood protein associated with the deficiency can increase circulating blood levels of the protein, thereby ameliorating or curing the disease. In one embodiment, the disease is hemophilia, e.g., hemophilia A or hemophilia B. In one embodiment, the gene is the F8 gene, which encodes clotting factor VIII. In one embodiment, the method includes knocking in the F8 gene, thereby treating or curing hemophilia A. In another embodiment, the gene is the F9 gene, which encodes clotting factor IX. In one embodiment, the method includes knocking in the F9 gene, thereby treating or curing hemophilia B. In one embodiment, the disease is A1AT deficiency. In one embodiment, the gene is the SERPINA1 gene, which encodes alpha-1-antitrypsin. In one embodiment, the method includes knocking in the SERPINA1 gene, thereby treating or preventing A1AT deficiency. In one embodiment, the disease is lysosomal acid lipase deficiency. In one embodiment, the gene is the LAL gene, which encodes lysosomal acid lipase, thereby treating or preventing lysosomal acid lipase deficiency.

[0219] In one embodiment, the disease is diabetes. In one embodiment, the gene encodes a secreted soluble blood protein. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in a gene encoding a secreted soluble blood protein, e.g., under the control of a druggable, inducible, or selectable promoter, can increase circulating blood levels of the protein and thereby ameliorate or cure the disease. In one embodiment, the gene is the INS gene, which encodes the protein insulin. In one embodiment, the gene is the GCG gene, which encodes the protein glucagon. In one embodiment, the method comprises knocking in the INS gene or the GCG gene, e.g., under the control of a druggable, inducible, or selectable promoter, thereby treating or preventing diabetes.

[0220] In one embodiment, the disease is growth hormone deficiency. In one embodiment, the gene is the GH gene, which encodes growth hormone. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in the GH gene, for example, under the control of a druggable, inducible, or selectable promoter, can increase circulating growth hormone levels, thereby improving or curing the disease. In one embodiment, the method includes knocking in the GH gene, for example, under the control of a druggable, inducible, or selectable promoter, thereby treating or preventing growth hormone deficiency.

[0221] In one embodiment, the disease is cancer, for example, a blood cancer. In one embodiment, the gene is a gene that is overexpressed in cancer. Without wishing to be bound by theory, in one embodiment, knocking down the gene, for example, by an eiCas9 molecule fused to a transcriptional repressor, is believed to ameliorate or cure the disease. In one embodiment, the gene is the EGFR gene. In one embodiment, the method includes activating the EGFR gene, thereby treating or preventing cancer progression and metastasis.

[0222] In one embodiment, the disease is hereditary hematoma. In one embodiment, the gene is a gene that is underexpressed in hereditary hematoma. Without wishing to be bound by theory, in one embodiment, it is believed that upregulating or activating the gene, for example, by an eiCas9 molecule fused to a transcriptional activator, improves or cures the disease. In one embodiment, the gene is the C1INH gene. In one embodiment, the method includes activating the C1INH gene, thereby treating or preventing hematoma.

[0223] In one embodiment, the disease is von Willebrand's disease. In one embodiment, the gene is underexpressed in von Willebrand's disease. Without wishing to be bound by theory, in one embodiment, it is believed that upregulation or activation of the gene, e.g., by an eiCas9 molecule fused to a transcriptional activator, ameliorates or cures the disease. In one embodiment, the gene is the VWF gene. In one embodiment, the method includes activating the VWF gene, thereby treating or preventing von Willebrand's disease.

[0224] In one embodiment, the disease is hereditary or acquired anemia. In one embodiment, the gene is a gene that is underexpressed in hereditary or acquired anemia. Without wishing to be bound by theory, in one embodiment, it is believed that transient upregulation or activation of the gene, for example, by an eiCas9 molecule fused to a transcriptional activator, improves or cures the disease. In one embodiment, the gene is the EPO gene. In one embodiment, the method includes a step of transiently activating the EPO gene, thereby treating or preventing hereditary or acquired anemia.

[0225] In one embodiment, the disease is neutropenia. In one embodiment, the gene is a gene that is underexpressed in neutropenia. Without wishing to be bound by theory, in one embodiment, it is believed that transient upregulation or activation of the gene, for example, by an eiCas9 molecule fused to a transcriptional activator, can ameliorate or cure the disease. In one embodiment, the gene is the CSF2 gene. In one embodiment, the method includes transiently activating the CSF2 gene, thereby treating or preventing neutropenia.

[0226] In one embodiment, the disease is a growth disorder. In one embodiment, the gene is a gene that is underexpressed in a growth disorder. Without wishing to be bound by theory, in one embodiment, it is believed that transient upregulation or activation of the gene, for example, by an eiCas9 molecule fused to a transcriptional activator, can ameliorate or cure the disease. In one embodiment, the gene is GH1. In one embodiment, the method includes transiently activating the GH1 gene, thereby treating or preventing the growth disorder.

[0227] In one embodiment, the disease is an infectious disease, an autoimmune disease, an inflammatory disease, a rheumatic disease, or a neoplastic disease. In one embodiment, the gene encodes a cytokine, a chemokine, an interleukin, or an inflammatory protein. Without wishing to be bound by theory, in one embodiment, it is believed that the disease can be ameliorated or cured by transiently or persistently downregulating or inhibiting a gene encoding a cytokine, a chemokine, an interleukin, or an inflammatory protein, for example, by an eiCas9 molecule fused to a transcriptional repressor (e.g., an inducible eiCas9 molecule). In one embodiment, the disease is a blood cancer. In one embodiment, the gene is the EPOR gene. In one embodiment, the method comprises knocking down the EPOR gene, thereby treating or preventing a blood cancer. In one embodiment, the disease is rheumatoid arthritis. In one embodiment, the gene is the TNF gene. In one embodiment, the method comprises knocking down the TNF gene, thereby treating or preventing rheumatoid arthritis. In one embodiment, the disease is an inflammatory disease. In one embodiment, the gene is the C5 gene. In one embodiment, the method comprises knocking down the C5 gene, thereby treating or preventing an inflammatory disease.

[0228] In one embodiment, the disease is an infectious disease, an autoimmune disease, an inflammatory disease, a rheumatic disease, or a neoplastic disease. In one embodiment, the gene encodes a cytokine, a chemokine, an interleukin, or an inflammatory protein. Without wishing to be bound by theory, in one embodiment, it is believed that the disease can be ameliorated or cured by transiently or persistently upregulating or activating a gene encoding a cytokine, a chemokine, an interleukin, or an inflammatory protein, for example, by an eiCas9 molecule fused to a transcriptional activator (e.g., an inducible eiCas9 molecule). In one embodiment, the disease is multiple sclerosis. In one embodiment, the gene is the IFNB1 gene. In one embodiment, the method includes activating the IFNB1 gene, thereby treating or preventing multiple sclerosis.

[0229] In one embodiment, the disease is an infectious disease, an autoimmune disease, an inflammatory disease, a rheumatic disease, or a neoplastic disease. In one embodiment, the gene encodes a cytokine, a chemokine, an interleukin, or an inflammatory protein receptor. Without wishing to be bound by theory, in one embodiment, knocking out a gene encoding a cytokine, a chemokine, an interleukin, or an inflammatory protein, for example, with an eaCas9 molecule, is believed to ameliorate or cure the disease. In one embodiment, the disease is HIV or AIDS. In one embodiment, the gene is CCR5. In another embodiment, the gene is the CXCR4 gene. In one embodiment, the method includes knocking out the CCR5 gene, the CXCR4 gene, or both, thereby treating or preventing HIV or AIDS.

[0230] In one embodiment, the disease is stroke or myocardial infarction. In one embodiment, the gene encodes a soluble blood protein, such as tissue plasminogen activator or urinary plasminogen activator. Without wishing to be bound by theory, in one embodiment, it is believed that upregulation or activation of the gene, e.g., transiently, e.g., by a transcriptionally fused eiCas9 molecule, can ameliorate or prevent the disease, e.g., prevent ischemia or dissolve blood clots. In one embodiment, the gene is the PLAT gene. In one embodiment, the method includes activating the PLAT gene, thereby treating or preventing stroke or myocardial infarction.

[0231] In one embodiment, the disease is a hemoglobinopathy. In one embodiment, the gene contains a mutation that causes a hemoglobinopathy. In one embodiment, the gene does not contain a mutation that causes a hemoglobinopathy. Without wishing to be bound by theory, in one embodiment, it is believed that knocking out or correcting the gene can ameliorate or cure the disease. In one embodiment, the gene containing the mutation is HBB, HBA1, or HBA2. In one embodiment, the method comprises correcting a mutated HBB, HBA1, or HBA2 gene, thereby treating or preventing sickle cell disease, alpha thalassemia, or beta thalassemia. In one embodiment, the gene is BCL11A. In one embodiment, the method comprises knocking out the BCL11A gene, thereby treating or preventing sickle cell disease or beta thalassemia.

[0232] In one embodiment, the disease is anemia. In one embodiment, the gene contains a mutation that causes anemia, for example, due to erythrocyte pyruvate kinase deficiency, such as hemolytic anemia. Without wishing to be bound by theory, in one embodiment, it is believed that anemia can be improved or cured by knocking in or correcting the gene. In one embodiment, the gene is PKLR. In one embodiment, the method includes correcting the knock-in in the wild-type PKLR gene or correcting the mutant PKLR gene, thereby treating or preventing anemia, for example, hemolytic anemia.

[0233] In one embodiment, the disease is a clotting factor disorder, e.g., hemophilia A. In one embodiment, the gene contains a mutation that causes the clotting factor disorder. Without wishing to be bound by theory, in one embodiment, it is believed that correcting the gene can ameliorate or cure the clotting factor disorder. In one embodiment, the gene is F8. In one embodiment, the method includes correcting a mutated F8 gene, thereby treating or preventing hemophilia A.

[0234] In one embodiment, the disease is a metabolic disorder, such as mucopolysaccharidosis type I. In one embodiment, the gene contains a mutation that causes the metabolic disorder. Without wishing to be bound by theory, it is believed that in one embodiment, knocking in or correcting the gene can improve or cure the metabolic disorder. In one embodiment, the gene is the IDUA gene. In one embodiment, the method involves knocking in a wild-type IDUA gene or correcting a mutant IDUA gene, thereby treating or preventing mucopolysaccharidosis type I.

[0235] In one embodiment, the disease is an immune deficiency, such as X-linked severe combined immunodeficiency. In one embodiment, the gene contains a mutation that causes the immune deficiency. Without wishing to be bound by theory, it is believed that in one embodiment, knocking in or correcting the gene can ameliorate or cure the disease. In one embodiment, the gene is the IL2RG gene. In one embodiment, the method involves knocking in a wild-type IL2RG gene or correcting a mutant IL2RG gene, thereby treating or preventing X-linked severe combined immunodeficiency.

[0236] In one embodiment, the disease is a myeloid immune deficiency, such as chronic granulomatous disease. In one embodiment, the gene contains a mutation that causes myeloid immune deficiency. Without wishing to be bound by theory, it is believed that in one embodiment, knocking in or correcting the gene can improve or cure the disease. In one embodiment, the gene is the NCF1 gene. In one embodiment, the method includes knocking in a wild-type NCF1 gene or correcting a mutant NCF1 gene, thereby treating or preventing chronic granulomatous disease.

[0237] In one embodiment, the disease is a beta lymphatic or immunoglobulin deficiency, e.g., X-linked agammaglobulinemia. In one embodiment, the gene contains a mutation associated with a beta lymphatic or immunoglobulin deficiency. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in or correcting the gene can ameliorate or cure the disease. In one embodiment, the gene is the BTK gene. In one embodiment, the method involves knocking in a wild-type BTK gene or correcting a mutant BTK gene, thereby treating or preventing X-linked agammaglobulinemia.

[0238] In one embodiment, the disease is cytopenia, for example, congenital amegakaryocytic thrombocytopenia type I. In one embodiment, the gene contains a mutation associated with cytopenia. Without wishing to be bound by theory, it is believed that in one embodiment, knocking in or correcting the gene can ameliorate or cure the disease. In one embodiment, the gene is the MPL gene. In one embodiment, the method involves knocking in a wild-type MPL gene or correcting a mutant MPL gene, thereby treating or preventing congenital amegakaryocytic thrombocytopenia type I.

[0239] In one embodiment, the disease is a metabolic disorder, enzyme deficiency, transport disorder, or storage disease, such as mucopolysaccharidosis IIIA. In one embodiment, the gene contains a mutation associated with the metabolic disorder, enzyme deficiency, transport disorder, or storage disease. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in or correcting the gene can improve or cure the disease. In one embodiment, the gene is the SGSH gene. In one embodiment, the method includes knocking in a wild-type SGSH gene or correcting a mutant SGSH gene, thereby treating or preventing mucopolysaccharidosis IIIA.

[0240] In one embodiment, the disease is an erythroid disease, such as primary familial congenital polycythemia. In one embodiment, the gene contains a mutation associated with the erythroid disease. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in or correcting the gene can improve or cure the disease. In one embodiment, the gene is the EPOR gene. In one embodiment, the method comprises knocking down the EPOR gene, either transiently or permanently, thereby treating or preventing primary familial congenital polycythemia.

[0241] In one embodiment, the disease is an erythroid disease, such as primary familial congenital polycythemia. In one embodiment, the gene contains a mutation associated with the erythroid disease. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in or correcting the gene can improve or cure the disease. In one embodiment, the gene is the EPOR gene. In one embodiment, the method includes knocking out or knocking down the EPOR gene, thereby treating or preventing primary familial congenital polycythemia.

[0242] Table 4 lists exemplary diseases that can be treated or prevented by the methods described herein and exemplary genes that can be modified by the methods described herein.

[0243] [Table 7]

[0244] [Table 8]

[0245] [Table 9]

[0246] [Table 10]

[0247] [Table 11]

[0248] [Table 12]

[0249] [Table 13]

[0250] [Table 14]

[0251] [Table 15]

[0252] In one embodiment, treatment is initiated in a subject after the onset of disease. In one embodiment, treatment is initiated in a subject after the onset of disease, but early in the course of disease progression (e.g., before the occurrence of specific symptoms), for example, to prevent disease progression. In one embodiment, the method comprises starting treatment in a subject at an advanced stage of disease, for example, to slow disease progression.

[0253] In one embodiment, the method is used to treat a subject having a disease described herein. In one embodiment, the method described herein is used to prevent or delay the onset or progression of a disease described herein.

[0254] In one embodiment, the method provides a selective advantage for the survival of one or more modified cells. In one embodiment, stem cells are modified to have a gene knockout, knock-in, knockdown, or correction. Unmodified diseased cells may undergo apoptosis. Thus, in one embodiment, after treatment as described herein, modified cells survive, while unmodified cells die. This selective advantage can drive the eventual formation of colonies of cells comprising at least 50%, e.g., at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% modified cells.

[0255] In one embodiment, the method includes initiating treatment in a subject who undergoes genetic testing that finds a mutation in a gene, e.g., a gene described herein.

[0256] In one embodiment, the method comprises initiating treatment in a subject who tests positive for a disease described herein.

[0257] In one embodiment, the method includes initiating treatment in a subject with a family history of the disease who exhibits any of the symptoms or signs of the disease and / or who is found to have a mutation in a gene associated with the disease.

[0258] In one embodiment, the method includes treating a subject who exhibits symptoms consistent with or associated with a disease.

[0259] In one embodiment, the method comprises isolating cells from a subject. In one embodiment, the cells are modified ex vivo and then returned (e.g., transplanted) to the subject. In one embodiment, the subject is the same subject from whom the cells were isolated. In another embodiment, the subject is a different subject from whom the cells were isolated. In one embodiment, autologous stem / progenitor cells are modified ex vivo and then returned to the subject. In another embodiment, xenogeneic stem / progenitor cells are modified ex vivo and then returned to the subject.

[0260] In one embodiment, the treatment involves delivery of a gRNA molecule, a Cas9 molecule, and optionally a donor template nucleic acid to a cell as described herein. In one embodiment, the gRNA molecule, the Cas9 molecule, or both, and optionally the donor template nucleic acid are delivered by a viral vector, e.g., an AAV vector or a lentiviral vector, e.g., an integrase-deficient lentivirus (IDLV). In another embodiment, the gRNA molecule and the Cas9 molecule are delivered as a gRNA molecule / Cas9 molecule ribonucleoprotein complex. In another embodiment, the gRNA molecule and the Cas9 molecule are delivered as RNA. In one embodiment, the template nucleic acid includes at least one exon of the target gene. In one embodiment, the template nucleic acid does not include a disease-associated mutation. In one embodiment, the template nucleic acid includes a promoter sequence. In another embodiment, the template nucleic acid does not include a promoter sequence. In one embodiment, the template nucleic acid includes a splice donor or acceptor. In another embodiment, the template nucleic acid includes a polyadenylation signal.

[0261] How to repair genetic mutations Disclosed herein is a method for modifying a target position (e.g., a target mutation position) in a gene, for example, a gene described herein. The modification of the target position can be achieved by repairing (e.g., correcting) one or more mutations in the gene, for example, by HDR. This approach corrects the mutant allele and returns it to a wild-type state. Without wishing to be bound by theory, in one embodiment, it is believed that correcting the mutation in the gene described herein restores wild-type gene activity. The method described herein can be carried out in any cell type, for example, the cell type described herein.

[0262] How to knock out or knock down a gene Disclosed herein are methods for modifying a target location (e.g., a target knockout or knockdown location) in a gene, for example, a gene described herein. The modification of the target location can be achieved, for example, by: (1) knocking out the gene: (a) inserting or deleting one or more nucleotides near or within the initial coding region of the gene (e.g., NHEJ-mediated insertion or deletion), or (b) deleting a genomic sequence containing at least a portion of the gene (e.g., NHEJ-mediated deletion), or (2) knocking down the gene mediated by an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., fused to a transcriptional repressor) by targeting the promoter region of the gene.

[0263] All techniques result in genetic modification. The methods described herein can be performed on any cell type, for example, the cell types described herein.

[0264] How to knock in a gene sequence Disclosed herein is a method for modifying a target position (e.g., a target knock-in position) in a gene, for example, a gene described herein. The modification of the target position can be achieved, for example, by knocking in a gene sequence, for example, a gene described herein (e.g., a cDNA encoding at least a portion of a gene described herein), for example, by HDR. Without wishing to be bound by theory, in one embodiment, it is believed that knocking in a gene sequence described herein restores wild-type gene activity. The methods described herein can be carried out in any cell type, for example, the cell types described herein.

[0265] How to activate genes Disclosed herein are methods for activating genes, such as those described herein. Gene activation can be mediated by an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., fused to a transcriptional activator) by targeting the promoter region of the gene. Without wishing to be bound by theory, in one embodiment, activation of the genes described herein is believed to restore wild-type gene activity. The methods described herein can be performed in any cell type, such as the cell types described herein.

[0266] Multiplexed modification of two or more genes The modification of two or more genes in the same cell or cells is referred to herein as "multiplexing." Multiplexing constitutes the modification of at least two genes in the same cell or cells.

[0267] For example, when two or more genes (e.g., CCR5 and CXCR4) are targeted for modification, the two or more genes (e.g., CCR5 and CXCR4) can be modified sequentially or simultaneously. In one embodiment, the CXCR4 gene is modified before the CCR5 gene. In one embodiment, the CXCR4 gene is modified simultaneously with the CCR5 gene. In one embodiment, the CXCR4 gene is modified subsequent to the CCR5 gene modification. In one embodiment, the effect of the modifications is synergistic. In one embodiment, the two or more genes (e.g., CCR5 and CXCR4) are modified sequentially to reduce the possibility of introducing a genomic rearrangement (e.g., a translocation) involving the two target locations.

[0268] II. Guide RNA (gRNA) molecules A gRNA molecule, as the term is used herein, is a nucleic acid that facilitates specific targeting or homing of a gRNA molecule / Cas9 molecule complex to a target nucleic acid. A gRNA molecule can be unimolecular (having a single RNA molecule), e.g., chimeric, or modular (comprising two or more, typically two separate RNA molecules). The gRNA molecules provided herein comprise a targeting domain that comprises, consists of, or consists essentially of a nucleic acid sequence that is fully or partially complementary to a target domain. In certain embodiments, the gRNA molecule further comprises one or more additional domains, including, for example, a first complementarity domain, a binding domain, a second complementarity domain, a flanking domain, a tail domain, and a 5' extension domain. This Each of these domains is described in further detail below. Further details about gRNAs are provided in Section I entitled "gRNA Molecules" of WO 2015 / 048577, the contents of which are expressly incorporated herein by reference. In certain embodiments, one or more domains in a gRNA molecule comprise an amino acid sequence identical to or having sequence homology to a naturally occurring sequence from, for example, S. pyogenes, S. aureus, or S. thermophilus.

[0269] In some embodiments, the unimolecular, or chimeric, gRNA preferably comprises, in 5' to 3' direction: a targeting domain complementary to a target domain in a gene, e.g., a gene described herein; a first complementarity domain; a binding domain; a second complementarity domain (complementary to the first complementarity domain); a flanking domain; and, optionally, a tail domain.

[0270] In some embodiments, the modular gRNA preferably comprises, in a 5' to 3' direction: a first strand comprising a targeting domain (complementary to the target domain in the gene; and a first complementarity domain; and a second strand preferably comprising, in a 5' to 3' direction: optionally, a 5' extension domain; a second complementarity domain; a flanking domain; and optionally, a tail domain.

[0271] Each of these domains is described in more detail below.

[0272] Targeted Domains A targeting domain (alternatively sometimes referred to as a guide sequence or complementary region) comprises, consists of, or consists essentially of a nucleic acid sequence that is complementary or partially complementary to a target nucleic acid sequence, e.g., a target nucleic acid sequence in a target gene. A nucleic acid sequence in a target gene, e.g., HBB, to which all or a portion of a targeting domain is complementary or partially complementary is referred to herein as a target domain. In certain embodiments, the target domain comprises a target location within a target gene, e.g., HBB. In other embodiments, the target location is outside of (i.e., upstream or downstream of) the target domain. In certain embodiments, the target domain is located entirely within the target gene, e.g., within a coding region, intron, or exon. In other embodiments, all or a portion of the target domain is located outside of the target gene, e.g., in a regulatory region or non-coding region.

[0273] Methods for selecting targeting domains are known in the art (see, e.g., Fu 2014; Sternberg 2014).

[0274] The strand of the target nucleic acid containing the targeting domain is referred to herein as the "complementary strand" because it is complementary to the targeting domain sequence. Because the targeting domain is part of the gRNA molecule, it contains the base uracil (U) rather than thymine (T); conversely, any DNA molecule encoding a gRNA molecule will contain thymine rather than uracil. In a targeting domain / targeting domain pair, the uracil base in the targeting domain will pair with the adenine base in the targeting domain. In certain embodiments, the degree of complementarity between the targeting domain and the targeting domain is sufficient to allow the Cas9 molecule to target the target nucleic acid.

[0275] In certain embodiments, the targeting domain comprises a core domain and an optional secondary domain. In some of these embodiments, the core domain is located 3' from the secondary domain, and in some of these embodiments, the core domain is located 3' from or near the 3' end of the targeting domain. In some of these embodiments, the core domain consists of, or consists essentially of, about 8 to about 13 nucleotides at the 3' end of the targeting domain. In certain embodiments, only the core domain is complementary or partially complementary to a corresponding portion of the targeting domain, and in some of these embodiments, the core domain is fully complementary to a corresponding portion of the targeting domain. In other embodiments, the secondary domain is also complementary or partially complementary to a portion of the targeting domain. In certain embodiments, the core domain is complementary or partially complementary to a core domain target in the targeting domain, while the secondary domain is complementary or partially complementary to a secondary domain target in the targeting domain. In certain embodiments, the core domain and the secondary domain have the same degree of complementarity as their respective corresponding portions of the targeting domain. In other embodiments, the degree of complementarity between the core domain and its target and the degree of complementarity between the secondary domain and its target may differ: in some of these embodiments, the core domain has a higher degree of complementarity to its target than the secondary domain, while in other embodiments, the secondary domain has a higher degree of complementarity than the core domain.

[0276] In certain embodiments, the targeting domain and / or core domain within the targeting domain is 3 to 100, 5 to 100, 10 to 100, or 20 to 100 nucleotides in length, and in some of these embodiments, the targeting domain or core domain is 3 to 15, 3 to 20, 5 to 20, 10 to 20, 15 to 20, 5 to 50, or 20 to 50 nucleotides in length. In certain embodiments, the targeting domain and / or core domain within the targeting domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certain embodiments, the targeting domain and / or the core domain within the targeting domain are 6+ / -2, 7+ / -2, 8+ / -2, 9+ / -2, 10+ / -2, 10+ / -4, 10+ / -5, 11+ / -2, 12+ / -2, 13+ / -2, 14+ / -2, 15+ / -2, or 16+ / -2, 20+ / -5, 30+ / -5, 40+ / -5, 50+ / -5, 60+ / -5, 70+ / -5, 80+ / -5, 90+ / -5, or 100+ / -5 nucleotides in length.

[0277] In certain embodiments where the targeting domain comprises a core domain, the core domain is 3 to 20 nucleotides in length; in some of these embodiments, the core domain is 5 to 15 or 8 to 13 nucleotides in length. In certain embodiments where the targeting domain comprises a secondary domain, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In certain embodiments where the targeting domain comprises a core domain that is 8 to 13 nucleotides in length, the targeting domain is 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, or 16 nucleotides in length, and the secondary domain is 13 to 18, 12 to 17, 11 to 16, 10 to 15, 9 to 14, 8 to 13, 7 to 12, 6 to 11, 5 to 10, 4 to 9, or 3 to 8 nucleotides in length.

[0278] In certain embodiments, the targeting domain is fully complementary to the target domain. Similarly, when the targeting domain comprises a core domain and / or a secondary domain, in certain embodiments, one or both of the core domain and the secondary domain are fully complementary to the corresponding portion of the target domain. In other embodiments, the targeting domain is partially complementary to the target domain, and in some of these embodiments in which the targeting domain comprises a core domain and / or a secondary domain, one or both of the core domain and the secondary domain are partially complementary to the corresponding portion of the target domain. In some of these embodiments, the nucleic acid sequence of the targeting domain, or the core domain or targeting domain within the targeting domain, is at least 80%, 85%, 90%, or 95% complementary to the target domain or the corresponding portion of the target domain. In certain embodiments, the targeting domain and / or a core or secondary domain within the targeting domain contains one or more nucleotides that are not complementary to the target domain or a portion thereof; in some of these embodiments, the targeting domain and / or a core or secondary domain within the targeting domain contains 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides that are not complementary to the target domain. In certain embodiments, the core domain contains 1, 2, 3, 4, or 5 nucleotides that are not complementary to the corresponding portion of the target domain. In certain embodiments, the targeting domain contains one or more nucleotides that are not complementary to the target domain, and the non-complementary nucleotide(s) is / are located within 5 nucleotides of the 5' or 3' end of the targeting domain. In some of these embodiments, the targeting domain contains 1, 2, 3, 4, or 5 nucleotides within 5 nucleotides of its 5' end, 3' end, or both its 5' and 3' ends that are not complementary to the target domain. In certain embodiments in which the targeting domain comprises two or more nucleotides that are not complementary to the targeting domain, two or more of the non-complementary nucleotides are adjacent to one another, and in some of these embodiments, the two or more contiguous non-complementary nucleotides are located within 5 nucleotides of the 5' or 3' end of the targeting domain.In other embodiments, the two or more consecutive non-complementary nucleotides are both located 6 or more nucleotides from the 5' and 3' ends of the targeting domain.

[0279] In one embodiment, the gRNA molecule is a modular gRNA molecule. In another embodiment, the gRNA molecule is a unimolecular or chimeric gRNA molecule.

[0280] In one embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, that includes a targeting domain that includes a sequence that is the same as or differs by 1, 2, 3, 4, or 5 or fewer nucleotides from the targeting domain sequence described herein. In one embodiment, the nucleic acid encodes a gRNA molecule that includes a targeting domain described herein.

[0281] In certain embodiments, the targeting domain comprises 16 nucleotides. In certain embodiments, the targeting domain comprises 17 nucleotides. In certain embodiments, the targeting domain comprises 18 nucleotides. In certain embodiments, the targeting domain comprises 19 nucleotides. In certain embodiments, the targeting domain comprises 20 nucleotides. In certain embodiments, the targeting domain comprises 21 nucleotides. In certain embodiments, the targeting domain comprises 22 nucleotides. In certain embodiments, the targeting domain comprises 23 nucleotides. In certain embodiments, the targeting domain comprises 24 nucleotides. In certain embodiments, the targeting domain comprises 25 nucleotides. In certain embodiments, the targeting domain comprises 26 nucleotides.

[0282] In certain embodiments, the targeting domain complementary to the gene is 16 or more nucleotides in length. In certain embodiments, the targeting domain is 16 nucleotides in length. In certain embodiments, the targeting domain is 17 nucleotides in length. In other embodiments, the targeting domain is 18 nucleotides in length. In yet other embodiments, the targeting domain is 19 nucleotides in length. In yet other embodiments, the targeting domain is 20 nucleotides in length. In yet other embodiments, the targeting domain is 21 nucleotides in length. In yet other embodiments, the targeting domain is 22 nucleotides in length. In yet other embodiments, the targeting domain is 23 nucleotides in length. In yet other embodiments, the targeting domain is 24 nucleotides in length. In yet other embodiments, the targeting domain is 25 nucleotides in length. In yet other embodiments, the targeting domain is 26 nucleotides in length.

[0283] In one embodiment, the nucleic acid encodes a modular gRNA molecule, e.g., one or more nucleic acids encode modular gRNA molecules. In another embodiment, the nucleic acid encodes a chimeric gRNA molecule. The nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain that is 16 nucleotides or more in length. In one embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain that is 16 nucleotides in length. In another embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain that is 17 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain that is 18 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain that is 19 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain that is 20 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain 21 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain 22 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain 23 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain 24 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain 25 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain 26 nucleotides in length.

[0284] In certain embodiments, the targeting domain, core domain, and / or secondary domain do not contain any modifications. In other embodiments, the targeting domain, core domain, and / or secondary domain, or one or more nucleotides therein, have modifications, including but not limited to those described below. In certain embodiments, one or more nucleotides of the targeting domain, core domain, and / or secondary domain may contain a 2' modification (e.g., a modification at the 2' position on the ribose), e.g., 2-acetylation, e.g., 2' methylation. In certain embodiments, the backbone of the targeting domain may be modified with phosphorothioate. In certain embodiments, modifications to one or more nucleotides of the targeting domain, core domain, and / or secondary domain render the targeting domain and / or gRNA comprising the targeting domain less susceptible to degradation or more biocompatible, e.g., less immunogenic. In certain embodiments, the targeting domain and / or core or secondary domain comprises 1, 2, 3, 4, 5, 6, 7, or 8 or more modifications, and in some of these embodiments, the targeting domain and / or core or secondary domain comprises 1, 2, 3, or 4 modifications within 5 nucleotides of their respective 5' ends and / or 1, 2, 3, or 4 modifications within 5 nucleotides of their respective 3' ends. In certain embodiments, the targeting domain and / or core or secondary domain comprises modifications at two or more consecutive nucleotides.

[0285] In certain embodiments where the targeting domain comprises a core and a secondary domain, the core and secondary domains comprise the same number of modifications. In some of these embodiments, neither domain comprises any modifications. In other embodiments, the core domain comprises more modifications than the secondary domain, or vice versa.

[0286] In certain embodiments, modifications to one or more nucleotides in the targeting domain, including within the core or secondary domain, are selected so as not to interfere with targeting efficiency, which can be evaluated by testing candidate modifications using the system described below. gRNAs having candidate targeting domains with selected lengths, sequences, degrees of complementarity, or degrees of modification can be evaluated using the system described below. Candidate targeting domains can be placed into a gRNA molecule / Cas9 molecule system known to be functional for a selected target and evaluated, either alone or together with one or more other candidate changes.

[0287] In certain embodiments, all of the modified nucleotides are complementary to and capable of hybridizing with corresponding nucleotides present in the target domain, while in other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 or more nucleotides are complementary to and capable of hybridizing with corresponding nucleotides present in the target domain.

[0288] First and second complementarity domains The first and second complementary domains (sometimes referred to as the crRNA-derived hairpin sequence and the tracrRNA-derived hairpin sequence, respectively) are fully or partially complementary to each other. In certain embodiments, the degree of complementarity is sufficient for the two domains to form a double-stranded region under at least some physiological conditions. In certain embodiments, the degree of complementarity between the first and second complementary domains is sufficient to enable targeting of the Cas9 molecule to a target nucleic acid, in conjunction with other properties of the gRNA.

[0289] In certain embodiments, the first and / or second complementary domains contain one or more nucleotides that lack complementarity with the corresponding complementary domain. In certain embodiments, the first and / or second complementary domains contain 1, 2, 3, 4, 5, or 6 nucleotides that are not complementary to the corresponding complementary domain. For example, the second complementary domain can contain 1, 2, 3, 4, 5, or 6 nucleotides that do not pair with the corresponding nucleotides in the first complementary domain. In some embodiments, the nucleotides in the first or second complementary domain that are not complementary to the corresponding complementary domain loop out from the duplex formed between the first and second complementary domains. In some of these embodiments, the unpaired loop out is located on the second complementary domain, and in some of these embodiments, the unpaired region begins at the 1st, 2nd, 3rd, 4th, 5th, or 6th nucleotide from the 5' end of the second complementary domain.

[0290] In certain embodiments, the first complementarity domain is 5 to 30, 5 to 25, 7 to 25, 5 to 24, 5 to 23, 7 to 22, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 7 to 15, 9 to 16, or 10 to 14 nucleotides in length, and in some of these embodiments, the first complementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In certain embodiments, the second complementarity domain is 5 to 27, 7 to 27, 7 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 7 to 20, 5 to 20, 7 to 18, 7 to 17, 9 to 16, or 10 to 14 nucleotides in length, and in some of these embodiments, the second complementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certain embodiments, the first and second complementarity domains are each independently 6+ / -2, 7+ / -2, 8+ / -2, 9+ / -2, 10+ / -2, 11+ / -2, 12+ / -2, 13+ / -2, 14+ / -2, 15+ / -2, 16+ / -2, 17+ / -2, 18+ / -2, 19+ / -2, or 20+ / -2, 21+ / -2, 22+ / -2, 23+ / -2, or 24+ / -2 nucleotides in length. In certain embodiments, the second complementarity domain is longer than the first complementarity domain, e.g., by 2, 3, 4, 5, or 6 nucleotides.

[0291] In certain embodiments, the first and / or second complementarity domains each independently comprise three subdomains, in the 5' to 3' direction: a 5' subdomain, a central subdomain, and a 3' subdomain. In certain embodiments, the 5' and 3' subdomains of the first complementarity domain are fully or partially complementary to the 3' and 5' subdomains, respectively, of the second complementarity domain.

[0292] In certain embodiments, the 5' subdomain of the first complementarity domain is 4 to 9 nucleotides in length, and in some of these embodiments, the 5' subdomain is 4, 5, 6, 7, 8, or 9 nucleotides in length. In certain embodiments, the 5' subdomain of the second complementarity domain is 3 to 25, 4 to 22, 4 to 18, or 4 to 10 nucleotides in length, and in some of these embodiments, the 5' domain is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In certain embodiments, the central subdomain of the first complementarity domain is 1, 2, or 3 nucleotides in length. In certain embodiments, the central subdomain of the second complementarity domain is 1, 2, 3, 4, or 5 nucleotides in length. In certain embodiments, the 3' subdomain of the first complementarity domain is 3 to 25, 4 to 22, 4 to 18, or 4 to 10 nucleotides in length, and in some of these embodiments, the 3' subdomain is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The subdomains are 4 to 9, eg, 4, 5, 6, 7, 8 or 9 nucleotides in length.

[0293] The first and / or second complementarity domains may share homology with or be derived from naturally occurring or reference first and / or second complementarity domains. In some of these embodiments, the first and / or second complementarity domains have at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% homology with the naturally occurring or reference first and / or second complementarity domains, or differ from them by no more than 1, 2, 3, 4, 5, or 6 nucleotides. In some of these embodiments, the first and / or second complementarity domains may have at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% homology with S. pyogenes or S. aureus.

[0294] In certain embodiments, the first and / or second complementarity domains do not contain any modifications. In other embodiments, the first and / or second complementarity domains or one or more nucleotides therein have modifications, including, but not limited to, those described below. In certain embodiments, one or more nucleotides of the first and / or second complementarity domains may contain a 2' modification (e.g., a modification at the 2' position on the ribose), e.g., 2-acetylation, e.g., 2-methylation. In certain embodiments, the backbone of the targeting domain may be modified with phosphorothioate. In certain embodiments, modifications to one or more nucleotides of the first and / or second complementarity domains render the first and / or second complementarity domains and / or gRNAs comprising the first and / or second complementarity domains less susceptible to degradation or more biocompatible, e.g., less immunogenic. In certain embodiments, the first and / or second complementarity domains comprise one, two, three, four, five, six, seven, or eight or more modifications, and in some of these embodiments, the first and / or second complementarity domains each independently comprise one, two, three, or four modifications within five nucleotides of their respective 5'-terminus, 3'-terminus, or both their respective 5'-terminus and 3'-terminus. In other embodiments, the first and / or second complementarity domains each independently comprise no modifications within five nucleotides of their respective 5'-terminus, 3'-terminus, or both their respective 5'-terminus and 3'-terminus. In certain embodiments, one or both of the first and second complementarity domains comprise modifications at two or more consecutive nucleotides.

[0295] In certain embodiments, modifications to one or more nucleotides in the first and / or second complementarity domains are selected so as not to interfere with targeting efficiency, which can be evaluated by testing candidate modifications using the system described below. gRNA molecules having candidate first or second complementarity domains with selected lengths, sequences, degrees of complementarity, or degrees of modification can be evaluated using the system described below. The candidate complementarity domains can be placed into a gRNA molecule / Cas9 molecule system known to be functional for the selected target and evaluated, either alone or together with one or more other candidate changes.

[0296] In certain embodiments, the duplex region formed by the first and second complementary domains is, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 base pairs in length (excluding any loop-out or unpaired nucleotides).

[0297] In certain embodiments, the first and second complementary domains, when duplexed, comprise 11 paired nucleotides (see, e.g., the gRNA of SEQ ID NO: 5). In certain embodiments, the first and second complementary domains, when duplexed, comprise 15 paired nucleotides (see, e.g., the gRNA of SEQ ID NO: 27). In certain embodiments, the first and second complementary domains, when duplexed, comprise 16 paired nucleotides (see, e.g., the gRNA of SEQ ID NO: 28). In certain embodiments, the first and second complementary domains, when duplexed, comprise 21 paired nucleotides (see, e.g., the gRNA of SEQ ID NO: 29).

[0298] In certain embodiments, one or more nucleotides are exchanged between the first and second complementary domains to remove the poly-U sequence. For example, nucleotides 23 and 48 or nucleotides 26 and 45 of a gRNA of SEQ ID NO:5 can be exchanged to generate a gRNA of SEQ ID NO:30 or 31, respectively. Similarly, nucleotides 23 and 39 of a gRNA of SEQ ID NO:29 can be exchanged with nucleotides 50 and 68 to generate a gRNA of SEQ ID NO:32.

[0299] Linked Domains In a unimolecular or chimeric gRNA, a linking domain is disposed between and serves to connect the first and second complementary domains. In certain embodiments, a portion of the linking domain is from a crRNA-derived region and another portion is from a tracrRNA-derived region.

[0300] In certain embodiments, the linking domain covalently links the first and second complementary domains. In certain embodiments, the linking domain consists of or comprises a covalent bond. In other embodiments, the linking domain non-covalently links the first and second complementary domains. In certain embodiments, the linking domain is 10 or fewer nucleotides in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In other embodiments, the linking domain is more than 10 nucleotides in length, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more nucleotides in length. In certain embodiments, the linking domain is between 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, 2 and 5, 10 and 100, 10 and 90, 10 and 80, 10 and 70, 10 and 60, 10 and 50, 10 and 40, 10 and 30, 10 and 20, 10 and 15, 20 and 100, 20 and 90, 20 and 80, 20 and 70, 20 and 60, 20 and 50, 20 and 40, 20 and 30, or 20 and 25 nucleotides in length. In certain embodiments, the linking domain is 10+ / -5, 20+ / -5, 30+ / -5, 30+ / -10, 40+ / -5, 40+ / -10, 50+ / -5, 50+ / -10, 60+ / -5, 60+ / -10, 70+ / -5, 70+ / -10, 80+ / -5, 80+ / -10, 90+ / -5, 90+ / -10, 100+ / -5, or 100+ / -10 nucleotides in length.

[0301] In certain embodiments, the linking domain shares homology with or is derived from a naturally occurring sequence, e.g., a sequence of the tracrRNA that is 5' to the second complementarity domain. In certain embodiments, the linking domain has at least 50%, 60%, 70%, 80%, 90%, or 95% homology to, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a linking domain described herein.

[0302] In certain embodiments, the linking domain does not contain any modifications. In other embodiments, the linking domain or one or more nucleotides therein have modifications, such as, but not limited to, those described below. In certain embodiments, one or more nucleotides of the linking domain may contain a 2' modification (e.g., a modification at the 2' position on the ribose), e.g., a 2-acetylation, e.g., a 2' methylation. In certain embodiments, the backbone of the linking domain can be modified with phosphorothioate. In certain embodiments, modifications to one or more nucleotides of the linking domain render the linking domain and / or gRNA comprising the linking domain less susceptible to degradation or more biocompatible, e.g., less immunogenic. In certain embodiments, the linking domain contains one, two, three, four, five, six, seven, or eight or more modifications; in some of these embodiments, the linking domain contains one, two, three, or four modifications within five nucleotides of its 5' and / or 3' end. In certain embodiments, the linking domain contains modifications at two or more consecutive nucleotides.

[0303] In certain embodiments, modifications to one or more nucleotides in the linking domain are selected so as not to interfere with targeting efficiency, which can be evaluated by testing the candidate modifications in the systems described below. gRNAs having candidate linking domains with selected lengths, sequences, degrees of complementarity, or degrees of modification can be evaluated in the systems described below. The candidate linking domains can be placed in a gRNA molecule / Cas9 molecule system known to be functional for the selected target and evaluated, either alone or together with one or more other candidate changes.

[0304] In certain embodiments, the linking domain typically comprises a duplex region adjacent to or within 1, 2, or 3 nucleotides of the 3' end of the first complementarity domain and / or the 5' end of the second complementarity domain. In some of these embodiments, the duplex region of the linking domain is 10 + / - 5, 15 + / - 5, 20 + / - 5, or 30 + / - 5 base pairs in length. In certain embodiments, the duplex region of the linking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length. In certain embodiments, the sequences forming the duplex region are fully complementary. In other embodiments, one or both of the sequences forming the duplex region contain one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides) that are not complementary to the other duplex sequence.

[0305] 5' extension domain In one embodiment, the modular gRNA disclosed herein comprises a 5' extension domain, i.e., one or more additional nucleotides 5' to the second complementarity domain. In certain embodiments, the 5' extension domain is 2-10 or more, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length, and in some of these embodiments, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

[0306] In certain embodiments, the 5' extension domain nucleotides do not include modifications, such as, for example, modifications of the type provided in Section VIII. However, in certain embodiments, the 5' extension domain includes one or more modifications, such as, for example, modifications that make it less susceptible to degradation or more biocompatible, such as, for example, less immunogenic. By way of example, the backbone of the 5' extension domain may be modified with phosphorothioate or other modifications described below. In certain embodiments, the nucleotides of the 5' extension domain may include a 2' modification (e.g., a modification at the 2' position on the ribose), such as, for example, a 2-acetylation, e.g., a 2' methylation, or other modifications described below.

[0307] In certain embodiments, the 5' extension domain may comprise as many as 1, 2, 3, 4, 5, 6, 7, or 8 modifications. In certain embodiments, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In certain embodiments, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.

[0308] In certain embodiments, the 5'-extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5'-end of the 5'-extension domain, within 5 nucleotides of the 3'-end of the 5'-extension domain, or more than 5 nucleotides away from one or both ends of the 5'-extension domain. In certain embodiments, two consecutive nucleotides that are within 5 nucleotides of the 5'-end of the 5'-extension domain, within 5 nucleotides of the 3'-end of the 5'-extension domain, or more than 5 nucleotides away from one or both ends of the 5'-extension domain are unmodified. In certain embodiments, nucleotides that are within 5 nucleotides of the 5'-end of the 5'-extension domain, within 5 nucleotides of the 3'-end of the 5'-extension domain, or more than 5 nucleotides away from one or both ends of the 5'-extension domain are unmodified.

[0309] Modifications in the 5' extension domain can be selected so as not to interfere with the gRNA molecule efficiency, which is evaluated by testing the candidate modifications in the system described in Section IV. gRNAs with candidate 5' extension domains of a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described below. Candidate 5' extension domains can be placed alone or together with one or more other candidate changes in a gRNA molecule / Cas9 molecule system known to be functional for a selected target and evaluated.

[0310] In certain embodiments, the 5' extension domain has at least 60, 70, 80, 85, 90, or 95% homology to, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference 5' extension domain, such as a 5' extension domain of natural origin, e.g., S. pyogenes, S. aureus, or S. thermophilus, and a 5' extension domain described herein.

[0311] Adjacent domains In certain embodiments, the flanking domain is 5 to 20 nucleotides in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In some of these embodiments, the flanking domain is 6 + / - 2, 7 + / - 2, 8 + / - 2, 9 + / - 2, 10 + / - 2, 11 + / - 2, 12 + / - 2, 13 + / - 2, 14 + / - 2, 14 + / - 2, 16 + / - 2, 17 + / - 2, 18 + / - 2, 19 + / - 2, or 20 + / - 2 nucleotides in length. In certain embodiments, the flanking domain is 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.

[0312] In certain embodiments, the flanking domains may share homology with or be derived from naturally occurring flanking domains. In some of these embodiments, the flanking domains share at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% homology with, or differ by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, the flanking domains disclosed herein, e.g., naturally occurring flanking domains such as S. pyogenes, S. aureus, or S. thermophilus.

[0313] In certain embodiments, the flanking domain does not contain any modifications. In other embodiments, the flanking domain or one or more nucleotides therein contains a modification, such as, but not limited to, the modifications described herein. In certain embodiments, one or more nucleotides of the flanking domain may contain a 2' modification (e.g., a modification at the 2' position on the ribose), e.g., a 2-acetylation, e.g., a 2' methylation. In certain embodiments, the backbone of the flanking domain can be modified with phosphorothioate. In certain embodiments, modifications to one or more nucleotides of the flanking domain render the flanking domain and / or gRNA comprising the flanking domain less susceptible to degradation or more biocompatible, e.g., less immunogenic. In certain embodiments, the flanking domain contains one, two, three, four, five, six, seven, or eight or more modifications; in some of these embodiments, the flanking domain contains one, two, three, or four modifications within five nucleotides of its 5' and / or 3' end. In certain embodiments, the flanking domain contains modifications at two or more consecutive nucleotides.

[0314] In certain embodiments, modifications to one or more nucleotides in the flanking domain are selected so as not to interfere with targeting efficiency, which can be evaluated by testing the candidate modifications in the systems described below. gRNAs having candidate flanking domains with selected lengths, sequences, degrees of complementarity, or degrees of modification can be evaluated in the systems described below. The candidate flanking domains can be placed in a gRNA molecule / Cas9 molecule system known to be functional for the selected target and evaluated, either alone or together with one or more other candidate changes.

[0315] Tail domain A variety of tail domains are suitable for use in the gRNA molecules disclosed herein.

[0316] In certain embodiments, the tail domain is absent. In other embodiments, the tail domain is 1 to 100 nucleotides in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length. In certain embodiments, the tail domain is 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 50, 10 to 100, 20 to 100, 10 to 90, 20 to 90, 10 to 80, 20 to 80, 10 to 70, 20 to 70, 10 to 60, 20 to 60, 10 to 50, 20 to 50, 10 to 40, 20 to 40, 10 to 30, 20 to 30, 20 to 25, 10 to 20, or 10 to 15 nucleotides in length. In certain embodiments, the tail domain is 5+ / -5, 10+ / -5, 20+ / -5, 25+ / -10, 30+ / -10, 30+ / -5, 40+ / -10, 40+ / -5, 50+ / -10, 50+ / -5, 60+ / -10, 60+ / -5, 70+ / -10, 70+ / -5, 80+ / -10, 80+ / -5, 90+ / -10, 90+ / -5, 100+ / -10 or 100+ / -5 nucleotides in length.

[0317] In certain embodiments, the tail domain may share homology with or be derived from a naturally occurring tail domain or the 5' end of a naturally occurring tail domain. In some of these embodiments, the flanking domain has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% homology to, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a naturally occurring tail domain disclosed herein, e.g., an S. pyogenes, S. aureus, or S. thermophilus tail domain.

[0318] In certain embodiments, the tail domains comprise sequences that are complementary to each other and form a duplex region under at least some physiological conditions. In some of these embodiments, the tail domain comprises a tail duplex domain that can form a tail duplex region. In certain embodiments, the tail duplex region is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In certain embodiments, the tail domain comprises a single-stranded domain 3' to the tail duplex domain that does not form a duplex. In some of these embodiments, the single-stranded domain is 3-10 nucleotides in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 4-6 nucleotides in length.

[0319] In certain embodiments, the tail domain does not contain any modifications. In other embodiments, the tail domain or one or more nucleotides therein contains a modification, such as, but not limited to, the modifications described herein. In certain embodiments, one or more nucleotides of the tail domain may contain a 2' modification (e.g., a modification at the 2' position on the ribose), e.g., a 2-acetylation, e.g., a 2' methylation. In certain embodiments, the backbone of the tail domain can be modified with phosphorothioate. In certain embodiments, modifications to one or more nucleotides of the tail domain render the tail domain and / or gRNA comprising the tail domain less susceptible to degradation or more biocompatible, e.g., less immunogenic. In certain embodiments, the tail domain contains one, two, three, four, five, six, seven, or eight or more modifications; in some of these embodiments, the tail domain contains one, two, three, or four modifications within five nucleotides of its 5' and / or 3' end. In certain embodiments, the tail domain contains modifications at two or more consecutive nucleotides.

[0320] In certain embodiments, modifications to one or more nucleotides in the tail domain are selected so as not to interfere with targeting efficiency, which can be evaluated by testing candidate modifications as described below. gRNAs with candidate tail domains of selected lengths, sequences, degrees of complementarity, or degrees of modification can be evaluated using the systems described below. Candidate tail domains can be placed in a gRNA molecule / Cas9 molecule system known to be functional for a selected target and evaluated, either alone or together with one or more other candidate changes.

[0321] In vivo or ex vivo transcription of gRNA In certain embodiments, the tail domain comprises the 3'-terminal nucleotides relevant to in vitro or in vivo transcription methods. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3'-end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the UUUUUU sequence. When an H1 promoter is used for transcription, these nucleotides may be the UUUU sequence. When an alternate Pol-III promoter is utilized, these nucleotides may be, for example, a variable number of uracil bases or may include alternate bases, depending on the termination signal of the Pol-III promoter. In certain embodiments, the flanking and tail domains together comprise, consist of, or consist essentially of the sequence set forth in SEQ ID NOs: 33, 34, 35, 36, or 38.

[0322] Assuming that T7 RNA polymerase requires G to initiate transcription, the T7 promoter typically has two Gs at its 3' end to ensure transcription of the entire RNA sequence downstream of the promoter. However, the resulting transcript may contain at least one G, if not both, from the promoter sequence, which may alter gRNA specificity or the interaction between the gRNA and the Cas9 protein. The gRNA target sequence begins with a G (e.g., HBB_8 gRNA target region DNA template: [ka] To address this concern, a new 5' sense primer [ka] (SEQ ID NO: 487, where the modified T7 promoter sequence is underlined) can be designed to remove two GGs from the T7 promoter sequence in the gRNA PCR template. A gRNA target sequence that does not start with G (e.g., HBB_15 gRNA target region DNA template: [ka] In this case, the T7 promoter sequence encoded in the gRNA PCR template can be modified so that only one of the Gs at the 3' end of the T7 promoter is removed: (Modified T7 promoter sequence: [ka] .

[0323] The T7 promoter sequence and modified T7 promoter sequence are not limited to those described herein. For example, the T7 promoter sequence (and modifications thereof) may be at least any of the sequences set forth in the Registry of Standard Biological Parts, "Promoters / Catalog / T7" (located at http: / / address:parts.igem.org / Promoters / Catalog / T7). The present disclosure provides that the gRNA disclosed herein is produced by in vitro transcription from a DNA template comprising a modified T7 promoter sequence described herein, in which one or more of the 3'-terminal Gs are removed (e.g., the sequence [ka] is located immediately upstream of a targeting domain lacking a G at its 5' end, or the sequence TAATACGACTCACTATA (SEQ ID NO:490) is located immediately upstream of a targeting domain with a G at its 5' end. Other modifications to these modified T7 promoters will be recognized by those of skill in the art based on other T7 promoter sequences, including at least any of the sequences set forth in the Registry of Standard Biological Parts, "Promoters / Catalog / T7" (located at http: / / address:parts.igem.org / Promoters / Catalog / T7, which is incorporated herein by reference in its entirety).

[0324] Exemplary Unimolecular / Chimeric gRNAs In certain embodiments, the gRNAs disclosed herein have the following structure: 5' [targeting domain]-[first complementary domain]-[linking domain]-[second complementary domain]-[flanking domain]-[tail domain]-3'; During the ceremony, the targeting domain comprises a core domain and optionally a secondary domain, and is 10 to 50 nucleotides in length; The first complementarity domain is 5-25 nucleotides in length and, in certain embodiments, has at least 50, 60, 70, 80, 85, 90, or 95% homology to a reference first complementarity domain disclosed herein; The linking domain is 1 to 5 nucleotides in length; The second complementarity domain is 5-27 nucleotides in length and, in certain embodiments, has at least 50, 60, 70, 80, 85, 90, or 95% homology to a reference second complementarity domain disclosed herein; The flanking domains are 5-20 nucleotides in length and, in certain embodiments, have at least 50, 60, 70, 80, 85, 90, or 95% homology to a reference flanking complementary domain disclosed herein; and The tail domain is absent or the nucleotide sequence is 1-50 nucleotides in length and, in certain embodiments, has at least 50, 60, 70, 80, 85, 90, or 95% homology to a reference tail domain disclosed herein.

[0325] In certain embodiments, the unimolecular gRNAs disclosed herein preferably comprise, in a 5' to 3' direction, the following: a targeting domain comprising, for example, 10 to 50 nucleotides; a first complementarity domain comprising, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides; a linking domain; a second complementarity domain; an adjacent domain; and a tail domain. During the ceremony, (a) the flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; (b) at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' of the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain.

[0326] In certain embodiments, the sequences from (a), (b), and / or (c) have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology to the corresponding sequence of a naturally occurring gRNA or to a gRNA described herein.

[0327] In certain embodiments, the flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0328] In certain embodiments, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

[0329] In certain embodiments, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' of the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain.

[0330] In certain embodiments, the targeting domain comprises, consists of, or consists essentially of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous nucleotides) that are complementary or partially complementary to the target domain or a portion thereof, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In some of these embodiments, the targeting domain is complementary to the target domain over the entire length of the targeting domain, the entire length of the targeting domain, or both.

[0331] In certain embodiments, a unimolecular or chimeric gRNA molecule disclosed herein (comprising a targeting domain, a first complementarity domain, a linking domain, a second complementarity domain, a flanking domain, and optionally, a tail domain) comprises the amino acid sequence set forth in SEQ ID NO:45, where the targeting domain is listed as 20 N (residues 1-20), but can range from 16 to 26 nucleotides in length, of which the last six residues (residues 97-102) represent the termination signal of the U6 promoter, but which may be absent or fewer. In certain embodiments, the unimolecular or chimeric gRNA molecule is a S. pyogenes gRNA molecule.

[0332] In certain embodiments, a unimolecular or chimeric gRNA molecule disclosed herein (comprising a targeting domain, a first complementarity domain, a linking domain, a second complementarity domain, a flanking domain, and optionally, a tail domain) comprises the amino acid sequence set forth in SEQ ID NO:40, where the targeting domain is listed as 20 N (residues 1-20), but it can range from 16 to 26 nucleotides in length, of which the last six residues (residues 97-102) represent the termination signal of the U6 promoter, but it can be absent or fewer. In certain embodiments, the unimolecular or chimeric gRNA molecule is a S. aureus gRNA molecule.

[0333] Exemplary modular gRNAs In certain embodiments, the modular gRNAs disclosed herein preferably comprise, in a 5' to 3' direction, the following: a targeting domain comprising, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides; a first strand comprising a first complementary domain; and, preferably, in a 5' to 3' direction, the following: optionally a 5' extension domain; a second complementary domain; a flanking domain; and a tail domain. During the ceremony, (a) the flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; (b) at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' of the last nucleotide of the second complementarity domain that is complementary to the corresponding nucleotide of the first complementarity domain.

[0334] In certain embodiments, the sequence from (a), (b), or (c) has at least 60, 75, 80, 85, 90, 95, or 99% homology to the corresponding sequence of a naturally occurring gRNA or to a gRNA described herein.

[0335] In certain embodiments, the flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0336] In certain embodiments, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0337] In certain embodiments, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain. In certain embodiments, the targeting domain comprises, has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) that have complementarity with the targeting domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.

[0338] In certain embodiments, the targeting domain comprises, consists of, or consists essentially of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous nucleotides) that are complementary to the target domain or a portion thereof. In some of these embodiments, the targeting domain is complementary to the target domain over the entire length of the target domain, the entire length of the target domain, or both.

[0339] In certain embodiments, the targeting domain comprises, has, or consists of 16 nucleotides (e.g., 16 consecutive nucleotides) that have complementarity with the targeting domain, e.g., the targeting domain is 16 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0340] In certain embodiments, the targeting domain comprises, has, or consists of 16 nucleotides (e.g., 16 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0341] In certain embodiments, the targeting domain comprises, has, or consists of 16 nucleotides (e.g., 16 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0342] In certain embodiments, the targeting domain has or consists of 17 nucleotides (e.g., 17 consecutive nucleotides) that are complementary to the target domain, e.g., the targeting domain is 17 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0343] In certain embodiments, the targeting domain has or consists of 17 nucleotides (e.g., 17 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0344] In certain embodiments, the targeting domain has or consists of 17 nucleotides (e.g., 17 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 17 nucleotides long. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0345] In certain embodiments, the targeting domain has or consists of 18 nucleotides (e.g., 18 consecutive nucleotides) that are complementary to the target domain, e.g., the targeting domain is 18 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0346] In certain embodiments, the targeting domain has or consists of 18 nucleotides (e.g., 18 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0347] In certain embodiments, the targeting domain has or consists of 18 nucleotides (e.g., 18 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 18 nucleotides long. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0348] In certain embodiments, the targeting domain comprises, has, or consists of 19 nucleotides (e.g., 19 contiguous nucleotides) that have complementarity with the targeting domain, e.g., the targeting domain is 19 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0349] In certain embodiments, the targeting domain comprises, has, or consists of 19 nucleotides (e.g., 19 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length. There are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0350] In certain embodiments, the targeting domain comprises, has, or consists of 19 nucleotides (e.g., 19 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0351] In certain embodiments, the targeting domain comprises, has, or consists of 20 nucleotides (e.g., 20 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0352] In certain embodiments, the targeting domain comprises, has, or consists of 20 nucleotides (e.g., 20 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0353] In certain embodiments, the targeting domain comprises, has, or consists of 20 nucleotides (e.g., 20 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0354] In certain embodiments, the targeting domain comprises, has, or consists of 21 nucleotides (e.g., 21 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0355] In certain embodiments, the targeting domain comprises, has, or consists of 21 nucleotides (e.g., 21 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0356] In certain embodiments, the targeting domain comprises, has, or consists of 21 nucleotides (e.g., 21 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0357] In certain embodiments, the targeting domain comprises, has, or consists of 22 nucleotides (e.g., 22 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0358] In certain embodiments, the targeting domain comprises, has, or consists of 22 nucleotides (e.g., 22 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0359] In certain embodiments, the targeting domain comprises, has, or consists of 22 nucleotides (e.g., 22 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0360] In certain embodiments, the targeting domain comprises, has, or consists of 23 nucleotides (e.g., 23 consecutive nucleotides) that have complementarity with the targeting domain, e.g., the targeting domain is 23 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0361] In certain embodiments, the targeting domain comprises, has, or consists of 23 nucleotides (e.g., 23 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0362] In certain embodiments, the targeting domain comprises, has, or consists of 23 nucleotides (e.g., 23 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0363] In certain embodiments, the targeting domain comprises, has, or consists of 24 nucleotides (e.g., 24 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0364] In certain embodiments, the targeting domain comprises, has, or consists of 24 nucleotides (e.g., 24 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0365] In certain embodiments, the targeting domain comprises, has, or consists of 24 nucleotides (e.g., 24 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0366] In certain embodiments, the targeting domain comprises, has, or consists of 25 nucleotides (e.g., 25 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0367] In certain embodiments, the targeting domain comprises, has, or consists of 25 nucleotides (e.g., 25 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length, and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementary domain.

[0368] In certain embodiments, the targeting domain comprises, has, or consists of 25 nucleotides (e.g., 25 consecutive nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 25 nucleotides long. There are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementary domain that is complementary to the corresponding nucleotide of the first complementary domain.

[0369] In certain embodiments, the targeting domain comprises, has, or consists of 26 nucleotides (e.g., 26 contiguous nucleotides) that have complementarity with the targeting domain, e.g., the targeting domain is 26 nucleotides in length. The flanking and tail domains together comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

[0370] In certain embodiments, the targeting domain comprises, has, or consists of 26 nucleotides (e.g., 26 contiguous nucleotides) that have complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length, and there are a...

Claims

1. 1. A method of producing modified cells for transplantation, comprising: (a) contacting the cells with a stem cell viability enhancer for a period of less than 120 hours; and then (b) contacting the cell with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer. The method includes:

2. 1. A method for modifying a target nucleic acid in a cell, comprising: stem cell viability enhancer; A modified gRNA molecule; and Cas9 molecule contacting the cells with The method includes:

3. 1. A method for transplanting modified cells into a subject, comprising: stem cell viability enhancer; A gRNA molecule; and Cas9 molecule to produce a modified cell; and transferring said modified cells into said subject. The method includes:

4. The contacting step comprises: (a) contacting the cells with the stem cell viability enhancer for a period of less than 120 hours; (b) contacting the cell with the gRNA molecule and the Cas9 molecule in the absence of the stem cell viability enhancer. The method of claim 2 or 3, comprising:

5. 5. The method of any one of claims 1 to 4, wherein the step of contacting the cell with the gRNA molecule and the Cas9 molecule is carried out using electroporation.

6. 6. The method of claim 5, further comprising subjecting the cells to cold shock prior to electroporation.

7. 7. The method of claim 5 or 6, further comprising the step of subjecting the cells to cold shock after electroporation.

8. The method of claim 6 or 7, wherein the cells are subjected to cold shock at a temperature of about 30°C to about 32°C.

9. 9. The method of any one of claims 4 to 8, wherein the period of less than 120 hours is about 72 hours.

10. 9. The method of any one of claims 1 or 4-8, wherein the period of less than 120 hours is about 24 to 48 hours.

11. 9. The method of any one of claims 1 or 4 to 8, wherein the period of less than 120 hours is a period that is not sufficiently long to promote expansion of the cells.

12. 12. The method of any one of claims 1 or 4-11, further comprising the step of: (c) contacting the cells with the stem cell viability enhancer for a period of less than 72 hours after step (b).

13. 13. The method of claim 12, wherein the period of less than 72 hours following step (b) is a period of time that is not sufficiently long to result in expansion of the cells.

14. 13. The method of claim 12, wherein the cell is a population of cells, and the number of cells in the population of cells does not increase by more than 5-fold in less than 72 hours following step (b).

15. 15. The method of claim 14, wherein the cell is a population of cells and the number of cells in the population of cells does not increase by more than two-fold in less than 72 hours following step (b).

16. 16. The method of any one of claims 12 to 15, wherein said period of less than 72 hours after step (b) is a period of about 24 to about 48 hours after step (b).

17. The method of any one of claims 1 to 16, wherein the cells are transferred into a human subject within 72 hours of completion of the contacting step.

18. 17. The method of any one of claims 1, 2, or 4-16, wherein the cells are cryopreserved within 72 hours of completion of the contacting step.

19. 19. The method of any one of claims 1 to 18, wherein said stem cell viability enhancer inhibits differentiation of said cell, inhibits its programmed cell death, inhibits its senescence, and inhibits its innate immune response.

20. 20. The method of claim 19, wherein the stem cell viability enhancer inhibits programmed cell death by inhibiting autophagy or apoptosis.

21. 20. The method of claim 3 or claim 17, wherein the cells engraft into a target tissue of the subject.

22. 22. The method of claim 21, wherein the cells comprise a population of cells, and at least 2% of the cells engraft into the target tissue of the subject.

23. 22. The method of claim 21, wherein the cells comprise a population of cells, and at least 15% of the cells engraft into the target tissue of the subject.

24. The method of any one of claims 21 to 23, wherein the cells remain engrafted in the target tissue of the subject for at least 16 weeks.

25. The method of any one of claims 21 to 24, wherein the target tissue is peripheral blood, bone marrow, or spleen.

26. The method of any one of claims 1 to 25, wherein the cells are stem cells.

27. 27. The method of claim 26, wherein the stem cells are hematopoietic stem / progenitor cells (HSCs).

28. 27. The method of any one of claims 1 to 26, wherein the cells are selected from the group consisting of circulating blood cells, mobilized blood cells, bone marrow cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, lineage-specific progenitor cells, endothelial cells, or mesenchymal stromal cells.

29. The method further comprising the step of isolating said cells from said subject prior to said contacting step.

30. 17. The method of any one of claims 1, 4, or 12-16, further comprising, after step (b), culturing the cells in a medium containing one or more cytokines.

31. 31. The method of claim 30, wherein the one or more cytokines are selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

32. 17. The method of any one of claims 1, 4, or 12-16, further comprising culturing the cells in a medium after step (b), wherein the medium comprises one or more of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-β signaling modulator, insulin-like growth factor binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), angiopoietin (ANG1), angiopoietin-like protein (ANGPTL4), an SDF1 / CXCR4 axis modulator, a Wnt signaling modulator, or a combination thereof.

33. 33. The method of any one of claims 30 to 32, wherein the cells are cultured in culture medium for at least 1, 2, 3, 4, 5, 6, or 7 days.

34. 34. The method of any one of claims 1 to 33, wherein the stem cell viability enhancer is an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist.

35. 35. The method of claim 34, wherein the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), LGC0006, α-naphthoflavone, and CH-223191.

36. 36. The method of claim 35, wherein the AhR antagonist is SR1.

37. 35. The method of claim 34, wherein the innate immune response antagonist is selected from the group consisting of cyclosporine A, dexamethasone, reservatrol, MyD88 inhibitory peptides, RNAi agents targeting Myd88, B18R recombinant protein, glucocorticoids, OxPAPC, TLR antagonists, rapamycin, BX795, and RLR shRNA.

38. 34. The method of any one of claims 1 to 33, wherein the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16,16-dimethyl prostaglandin E2 (dmPGE2).

39. 39. The method of any one of claims 1-38, further comprising contacting the cell with a second Cas9 molecule.

40. The method of any one of claims 1 to 39, wherein the Cas9 molecule is an enzymatically active Cas9 (eaCas9).

41. 41. The method of claim 40, wherein the Cas9 molecule is capable of catalyzing a single-stranded cleavage in a target nucleic acid.

42. 41. The method of Claim 40, wherein the Cas9 molecule is capable of catalyzing a double-stranded break in a target nucleic acid.

43. 40. The method of any one of claims 1 to 39, wherein the Cas9 molecule is selected from the group consisting of wild-type Cas9, nickase Cas9, inactive Cas9 (dCas9), split Cas9, and inducible Cas9.

44. The method of any one of claims 1 to 41, wherein the Cas9 molecule comprises N-terminal RuvC-like domain cleavage activity but not HNH-like domain cleavage activity.

45. 45. The method of claim 44, wherein the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position N863 of Streptococcus pyogenes Cas9.

46. 42. The method of any one of claims 1 to 41, wherein the Cas9 molecule comprises HNH-like domain cleavage activity but does not have N-terminal RuvC-like domain cleavage activity.

47. 47. The method of claim 46, wherein the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

48. The method of any one of claims 1 to 47, wherein the Cas9 molecule is a Cas9 polypeptide.

49. 49. The method of claim 48, wherein the Cas9 polypeptide is a Staphylococcus aureus Cas9 polypeptide.

50. 49. The method of claim 48, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

51. 51. The method of any one of claims 48-50, wherein the gRNA molecule and the Cas9 polypeptide are linked in a preformed ribonucleotide complex.

52. The method of any one of claims 1 to 47, wherein the Cas9 molecule is a nucleic acid encoding a Cas9 polypeptide.

53. The method of any one of claims 1 to 52, wherein the gRNA molecule is a modified gRNA molecule.

54. 54. The method of Claim 53, wherein the modified gRNA molecule comprises a 5'-end cap structure.

55. The method of claim 53 or claim 54, wherein the repair gRNA molecule comprises a 3'-terminal poly-A tail.

56. 56. The method of any one of claims 1 to 55, further comprising contacting the cell with a template nucleic acid.

57. 57. The method of claim 56, wherein the template nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

58. 57. The method of claim 56, wherein the template nucleic acid is present in an adeno-associated virus (AAV) or an integration defective virus (ILDV).

59. 58. The method of claim 57, wherein the ssODN comprises a 5' phosphorothioate modification.

60. 58. The method of claim 57, wherein the ssODN comprises a 3' phosphorothioate modification.

61. 58. The method of claim 57, wherein the ssODN comprises a 5' phosphorothioate modification and a 3' phosphorothioate modification.

62. 62. The method of any one of claims 1 to 61, further comprising contacting the cell with a transgene, wherein the contacting step is performed under conditions that allow the transgene to be integrated into the genome of the stem cell.

63. 63. The method of claim 62, wherein the transgene further comprises a promoter and a poly-adenine tail.

64. 63. The method of claim 62, wherein the transgene is a gene that is a chemotherapy selection marker, a cell surface antigen, or a suicide gene.

65. 65. The method of any one of claims 1-64, further comprising contacting the cell with an eiCas9 molecule.

66. 66. The method of claim 65, wherein the eiCas9 molecule is fused to a transcriptional repressor or a transcriptional activator.

67. 67. The method of any one of claims 1 to 66, wherein the gRNA molecule comprises a targeting domain complementary to a target domain in a target gene.

68. 68. The method of claim 67, wherein the target gene is listed in Table 4.

69. 1. A method for generating hematopoietic stem / progenitor cells (HSCs) for transplantation, comprising: (a) contacting the HSCs with a stem cell viability enhancer for a period of less than 72 hours; and then (b) electroporating the HSCs with a modified gRNA molecule and a Cas9 polypeptide in the absence of the stem cell viability enhancer, wherein the modified gRNA molecule and the Cas9 polypeptide are associated in a preformed ribonucleotide complex, and the modified gRNA molecule comprises a 5' end modification comprising a 3'-O-Me-m7G(5')ppp(5')G anti-reverse cap analog (ARCA) and a 3' end modification comprising a polyadenine tail.

70. 70. The method of claim 69, further comprising contacting the HSCs during step (a) with one or more cytokines selected from the group consisting of: stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

71. 71. The method of claim 69 or claim 70, wherein the stem cell viability enhancer is SR1, dmPGE2, or a combination of SR1 and dmPGE2.

72. 72. The method of any one of claims 69-71, wherein the Cas9 polypeptide is a wild-type Cas9 polypeptide or a Cas9 polypeptide comprising an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

73. 72. The method of any one of claims 69 to 71, further comprising a step of subjecting the cell to a cold shock after electroporation.

74. A cell modified by the method of any one of claims 1 to 73.

75. A pharmaceutical composition comprising the cells of claim 74.

76. 74. A method of treating or preventing a disease in a subject, comprising the step of administering to said subject modified cells or cells altered by the method of any one of claims 1 to 73.

77. stem cell viability enhancer; A modified gRNA molecule; and Cas9 molecule A stem cell gene editing system comprising:

78. 78. The stem cell gene editing system of Claim 77, further comprising a stem cell, wherein said stem cell comprises said modified gRNA molecule and said Cas9 molecule.

79. 78. The stem cell gene editing system of Claim 77, further comprising a stem cell, wherein the stem cell comprises a stem cell viability enhancer.

80. The stem cell gene editing system of any one of claims 77 to 79, wherein the stem cell viability enhancer is selected from the group consisting of an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist.

81. The stem cell gene editing system of claim 80, wherein the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), AhRA, dimethoxyflavone, 6,2',4'-trimethoxyflavone, LGC0006, α-naphthoflavone, and CH-223191.

82. The stem cell gene editing system of claim 81, wherein the AhR antagonist is SR1.

83. The stem cell gene editing system of claim 80, wherein the innate immune response antagonist is selected from the group consisting of cyclosporine A, dexamethasone, resveratrol, MyD88 inhibitory peptides, RNAi agents targeting Myd88, B18R recombinant protein, glucocorticoids, OxPAPC, TLR antagonists, rapamycin, BX795, and RLR inhibitors.

84. The stem cell gene editing system of any one of claims 77 to 79, wherein the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16,16-dimethyl prostaglandin E2 (dmPGE2).

85. The stem cell gene editing system of any one of claims 77 to 84, wherein the modified gRNA molecule comprises a 5'-end cap structure.

86. The stem cell gene editing system of any one of claims 77 to 85, wherein the modified gRNA molecule comprises a 3'-terminal polyadenine tail.

87. The stem cell gene editing system of any one of claims 77 to 86, further comprising a second Cas9 molecule.

88. The stem cell gene editing system of any one of claims 77 to 87, wherein the Cas9 molecule is enzymatically active Cas9 (eaCas9).

89. The stem cell gene editing system of claim 88, wherein the eaCas9 molecule is capable of catalyzing a single-stranded break in a target nucleic acid.

90. The stem cell gene editing system of claim 88, wherein the eaCas9 is capable of catalyzing a double-stranded break in the target nucleic acid.

91. The stem cell gene editing system of any one of claims 77 to 90, wherein the Cas9 molecule is selected from the group consisting of wild-type Cas9, nickase Cas9, inactive Cas9 (dCas9), split Cas9, and inducible Cas9.

92. The stem cell gene editing system of any one of claims 77 to 89, wherein the Cas9 molecule comprises an N-terminal RuvC-like domain cleavage activity but does not have HNH-like domain cleavage activity.

93. The stem cell gene editing system of Claim 92, wherein the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position N863 of Streptococcus pyogenes Cas9.

94. The stem cell gene editing system of any one of claims 77 to 89, wherein the Cas9 molecule comprises HNH-like domain cleavage activity but does not have N-terminal RuvC-like domain cleavage activity.

95. The stem cell gene editing system of Claim 94, wherein the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

96. The stem cell gene editing system of any one of claims 77 to 95, wherein the Cas9 molecule is a Cas9 polypeptide.

97. The stem cell gene editing system of claim 96, wherein the Cas9 polypeptide is a Staphylococcus aureus Cas9 polypeptide.

98. The stem cell gene editing system of claim 96, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

99. The stem cell gene editing system of any one of claims 96 to 98, wherein the gRNA molecule and the Cas9 polypeptide are linked in a preformed ribonucleotide complex.

100. The stem cell gene editing system of any one of claims 77 to 95, wherein the Cas9 molecule is a nucleic acid encoding a Cas9 polypeptide.

101. A stem cell gene editing system described in any one of claims 77 to 100, further comprising a cytokine.

102. The stem cell gene editing system of claim 101, wherein the cytokine is selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

103. 103. The stem cell gene editing system of any one of claims 77-102, further comprising one or more of the following: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-β signaling modulator, insulin-like growth factor binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), angiopoietin (ANG1), angiopoietin-like protein (ANGPTL4), an SDF1 / CXCR4 axis modulator, or a Wnt signaling modulator.

104. The method of any one of claims 77 to 103, further comprising a template nucleic acid.

105. The stem cell gene editing system of claim 104, wherein the template nucleic acid is a single-stranded oligodeoxynucleotide (ssODN).

106. The stem cell gene editing system of claim 104, wherein the template nucleic acid is present in an adeno-associated virus (AAV) or an integration defective virus (ILDV).

107. The stem cell gene editing system of claim 105, wherein the ssODN comprises a 5' phosphorothioate modification, a 3' phosphorothioate modification, or both a 5' phosphorothioate modification and a 3' phosphorothioate modification.

108. The stem cell gene editing system of any one of claims 77 to 107, wherein the gRNA molecule comprises a targeting domain complementary to a target domain in a target gene.

109. The stem cell gene editing system of claim 108, wherein the target gene is listed in Table 4.

110. A stem cell comprising a modification in a target nucleic acid, wherein the stem cell is (a) contacted with a stem cell viability enhancer for a period of less than 120 hours, and then (b) contacted with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer.

111. A stem cell comprising a modification in a target nucleic acid according to claim 110, wherein the stem cell is (c) further contacted with the stem cell viability enhancer for a period of less than 72 hours after step (b).

112. 111. A stem cell comprising a modification in a target nucleic acid according to claim 110, wherein the period of less than 72 hours following step (b) is a period not sufficiently long to result in expansion of the stem cell.

113. A population of stem cells comprising a modification in a target nucleic acid, wherein the population of stem cells is (a) contacted with a stem cell viability enhancer for a period of less than 120 hours, and then (b) contacted with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer.

114. 114. The population of stem cells of claim 113, wherein the population of stem cells is (c) further contacted with the stem cell viability enhancer for a period of less than 72 hours after step (b).

115. 114. The population of stem cells of claim 113, wherein the period of less than 72 hours following step (b) is a period not long enough to result in expansion of the stem cells.

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