Compositions and methods for treatment of hemoglobinopathies

Abstract

To provide genome editing systems, reagents and methods for the treatment of hemoglobinopathies.SOLUTION: Provided is a gRNA molecule comprising a tracr and crRNA, wherein the crRNA comprises a targeting domain that is complementary with a target sequence within a specific genomic nucleic acid sequence, is complementary with a target sequence of a nondeletional HFPH region (e.g., a human nondeletional HPFH region), or combinations thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a direct sequel to U.S. Provisional Patent Application No. 62 / 455,464, filed February 6, 2017. No. 60 / 699,999, filed on Oct. 1, 2003, entitled "Compounds for the Prevention of Violation of Regulation (EU) No. 60 / 699,999, filed on Oct. 1, 2003, and ... will be done.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format, the entirety of which is hereby incorporated by reference. The ASCII copy, created on January 30, 2018, is incorporated herein by reference. The file is named AT057603-WO-PCT_SL.txt and contains 258,837 bytes. The size of the [Background technology]

[0003] CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats (clustered regularly spaced repeats) The short palindromic repeats of )) are a type of nucleotide that acts as a defense against viral attack in bacteria. It evolved as an adaptive immune system. Upon exposure to a virus, it binds to short segments of viral DNA. The CRISPR gene containing the viral sequence is integrated into the CRISPR locus in the bacterial genome. RNA is transcribed from a portion of the R locus. This R contains a sequence complementary to the viral genome. The NA mediates targeting of the Cas9 protein to sequences in the viral genome. The as9 protein cleaves and thereby silences the viral target.

[0004] In recent years, the CRISPR / Cas system has been applied to genome editing in eukaryotic cells. By introducing site-specific single-strand breaks (SSBs) or double-strand breaks (DSBs), For example, non-homologous end joining (NHEJ) or homology-directed repair (HDR) can be used to modify the target sequence. It becomes possible to change. Summary of the Invention [Means for solving the problem]

[0005] Without being bound by theory, the present invention is based in part on the using a CRISPR system, e.g., a Cas9 CRISPR system, as described in thereby resulting in the development of fetal hemoglobin in, for example, progeny of the modified cells, e.g., red blood cell progeny. increased β-globin (HbF) expression and / or β-globin (e.g., those with disease-causing mutations) globin gene, which is involved in the expression of the β-globin gene, is reduced, for example, by the use of a non- Modifying cells (e.g., hematopoietic stem and progenitor cells (HSPCs)) in the deleted HPFH region By using modified cells (e.g., modified HSPCs), abnormal hemoglobin can be reduced. This is based on the discovery that globinopathies, such as sickle cell disease and beta thalassemia, can be treated. In embodiments, targeting regions of the genome where the HPFH mutation or deletion map is unknown, A gene editing system, e.g., as described herein, is administered to a cell (e.g., an HSPC). For example, by introducing the CRISPR system, it is possible to efficiently engraft the cells into living organisms. persists for a long time in culture (including differentiation into erythroid cells with increased fetal hemoglobin expression) modified HSPCs (e.g., as described herein) that are capable of differentiation It was surprisingly shown that HSPCs containing one or more indels in These modified HSPCs are then grown under conditions that promote stem cell proliferation and expansion while maintaining stemness. For example, in the presence of a stem cell proliferation agent (e.g., as described herein), They can be cultured in vivo, e.g., for gene editing as described herein. Systems, such as the CRISPR system, have been introduced into HPSCs derived from sickle cell disease patients. When the modified cells and their progeny (e.g., erythroid progeny) are introduced into the embryo, surprisingly, Not only did they show upregulation of fetal hemoglobin, but also sickle beta-globin expression relative to unmodified cell populations. There is also a significant decrease in the number of sickle cells and an increase in the number of normal red blood cells.

[0006] Thus, in one embodiment, the present invention provides a method for producing a gRNA molecule as described herein. A CRISPR system (e.g., a Cas CRISPR system) containing one or more, e.g., one, stem, e.g., Cas9 CRISPR system, e.g., Streptococcus pyogenes (S. pyog) Cas9 CRISPR systems are provided. The methods and cells described herein may use any of the gRNA molecules described herein. obtain.

[0007] In one embodiment, the present invention provides a gRNA molecule comprising tracr and crRNA. , where crRNA is a) complementary to a target sequence in a non-deleted HFPH region (e.g., a human non-deleted HFPH region); ; b)Chr11:5,249,833~Chr11:5,250,237, -chain, hg3 8, which is complementary to a target sequence within the genomic nucleic acid sequence; c)Chr11:5,254,738~Chr11:5,255,164, -chain, hg3 8, which is complementary to a target sequence within the genomic nucleic acid sequence; d) Chr11:5,250,094-5,250,237, -strand, genomic DNA fragment in hg38 is complementary to a target sequence within the nucleic acid sequence; e) Chr11:5,255,022-5,255,164, -strand, genomic DNA fragment in hg38 is complementary to a target sequence within the nucleic acid sequence; f) Chr11:5,249,833-5,249,927, -strand, genomic DNA fragment in hg38 is complementary to a target sequence within the nucleic acid sequence; g) Chr11:5,254,738-5,254,851, -strand, genomic DNA in hg38 is complementary to a target sequence within the nucleic acid sequence; h) Chr11:5,250,139-5,250,237, -strand, genomic DNA fragment in hg38 is complementary to a target sequence within the nucleic acid sequence; or i) combinations of these The targeting domain is

[0008] In embodiments, the targeting domain comprises any one of SEQ ID NO: 1 to SEQ ID NO: 72. In embodiments, the targeting domain comprises, for example, consists of, SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:2 8, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, The present invention may further comprise, for example, any one of SEQ ID NO: 63 or SEQ ID NO: 67. wherein the targeting domain is a) SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO: 48, SEQ ID NO: 51 or SEQ ID NO: 67; or b) SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 54, e.g. In embodiments, the gRNA molecule comprises, e.g., consists of, SEQ ID NO: 8. In an embodiment, the gRNA molecule comprises a targeting domain consisting of SEQ ID NO: 67. In embodiments, the gRNA molecule comprises a targeting domain comprising, for example, The targeting domain comprises (e.g., consists of) a fragment of any of the sequences of

[0009] In any of the foregoing aspects and embodiments, the gRNA molecule may be any of the gRNA molecules described herein. In embodiments, the gRNA molecule may further comprise a target In embodiments, the targeting domain comprises any fragment of the targeting domain described herein. comprising 17, 18, 19, or 20 contiguous nucleic acids of any one of the targeting domain sequences In embodiments, any one of the targeting domain sequences described herein may be selected from the group consisting of: The 17, 18, 19, or 20 contiguous nucleic acids are 3 or 4 of the targeting domain sequence described. In other embodiments, the nucleic acid sequence is 17, 18, 19, or 20 consecutive nucleic acids arranged at the termini. 17, 18, 19, or 20 of any one of the targeting domain sequences described The contiguous nucleic acid is located at the 5' end of the targeting domain sequence described. In other embodiments, the targeting domain is 9 or 20 contiguous nucleic acids. Any one of 17, 18, 19, or 20 contiguous nucleic acids of the sequence may be a targeting sequence as described. In an embodiment, the targeting domain does not include any of the nucleic acids 5' or 3' of the targeting domain sequence. The domain consists of the targeting domain sequence described.

[0010] In certain aspects, including in any of the foregoing aspects and embodiments, A portion of the tracr hybridizes to include SEQ ID NO: 182 or 183.

[0023] The present invention provides a flagpole including any of the above aspects and embodiments. In some embodiments, the flagpole is located 3' to the crRNA portion of the flagpole. and a first flagpole extension having an arrangement In some aspects, including in any of the above aspects and embodiments, The flagpole is a second flagpole located 3' to the crRNA portion of the flagpole. further comprising a drag pole extension and, if present, a first flag pole extension, The second flagpole extension comprises SEQ ID NO:185.

[0011] In certain aspects, including in any of the foregoing aspects and embodiments, tracr In any of the foregoing aspects and embodiments, the nucleotide sequence comprises SEQ ID NO: 224 or SEQ ID NO: 225. In some embodiments, the tracr may optionally include an additional 1, 2, 3, 4 SEQ ID NO: 232, further comprising 5, 6 or 7 uracil (U) nucleotides at the 3' end In certain aspects, including in any of the above aspects and embodiments, The RNA contained, from 5' to 3', [targeting domain]-: a) SEQ ID NO: 182; b) SEQ ID NO: 183; c) SEQ ID NO: 199; d) SEQ ID NO: 200; e) SEQ ID NO: 201; f) SEQ ID NO: 202; or g) comprising SEQ ID NO: 226.

[0012] In certain aspects, including in any of the foregoing aspects and embodiments, tracr is, from 5' to 3', a) SEQ ID NO: 187; b) SEQ ID NO: 188; c) SEQ ID NO: 203; d) SEQ ID NO: 204; e) SEQ ID NO: 224; f) SEQ ID NO: 225; g) SEQ ID NO: 232; h) SEQ ID NO: 227; i) (SEQ ID NO: 228; j) SEQ ID NO: 229; k) at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g., 1, 2, 3, 4, 5 Any of the above a) to j) further containing 6 or 7 uracil (U) nucleotides at the 3' end. l) at least 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides For example, 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides are attached to the 3' end. any of the above a) to k); or m) at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, such as 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides A) Any of the above a) to l) further containing a nucleotide at the 5' end (e.g., at the 5' end) Including Reka.

[0013] In certain aspects, including in any of the aforementioned aspects and embodiments, a targeting domain In any of the foregoing aspects and embodiments, the indole and tracr are located on separate nucleic acid molecules. In some embodiments, including those described below, the targeting domain and the tracr are located in separate nuclei. The nucleic acid molecule, which is disposed on the nucleic acid molecule and comprises a targeting domain, optionally comprises all of the targeting domain. and a nucleic acid molecule comprising SEQ ID NO: 201 located immediately 3' to the tracr sequence. 224, including, for example, consisting of, any of the above aspects and embodiments. Therefore, in one embodiment, the crRNA portion of flagpole is SEQ ID NO: 201 or SEQ ID NO: In some aspects, including in any of the above aspects and embodiments, tracr is a sequence of SEQ ID NO: 187 or 188 and optionally a first flagpole extension If present, the first tracr extension located 5' of SEQ ID NO: 187 or 188 The first tracr extension comprises SEQ ID NO:189.

[0014] In certain aspects, including in any of the aforementioned aspects and embodiments, a targeting domain The indole and tracr are located on a single nucleic acid molecule, e.g., tracr is a targeting domain. In some embodiments, the gRNA molecule is positioned 3' of the targeting domain. In an embodiment, the loop comprises a loop located 5' to the nucleotide sequence of the tracr gene. In some aspects, including in any of the above aspects and embodiments, wherein the gRNA molecule comprises, from 5' to 3', [targeting domain]-: (a) SEQ ID NO: 19 5; (b) SEQ ID NO: 196; (c) SEQ ID NO: 197; (d) SEQ ID NO: 198; (e) SEQ ID NO: No. 231; or (f) 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides at the 3' end.

[0015] In certain aspects, including in any of the aforementioned aspects and embodiments, a targeting domain The targeting domain and the tracr are located on a single nucleic acid molecule, and the nucleic acid molecule and optionally SEQ ID NO: 231 located immediately 3' of the targeting domain. , for example consisting of

[0016] In certain aspects, including in any of the foregoing aspects and embodiments, One or optionally more of the nucleic acid molecules comprising the nucleic acid sequence a) one or more, e.g., three, phosphorothioates at the 3' end of said one or more nucleic acid molecules Oate modification; b) one or more, e.g., three, phosphorothioates at the 5' end of said one or more nucleic acid molecules Oate modification; c) one or more, for example, three 2'-O- methyl modification; d) one or more, for example, three 2'-O- methyl modification; e) the fourth, third and second terminal ends of said one or more nucleic acid molecules 2'O-methyl modifications at each of the 3' residues of; f) the fourth, third and second terminal ends of said one or more nucleic acid molecules a 2'O-methyl modification at each of the 5' residues of f) Any combination of these Includes:

[0017] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 74; (b) SEQ ID NO: 75; or (c) SEQ ID NO: 76 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0018] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 77 and comprising SEQ ID NO: 224; For example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 77 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, it consists of tracr; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 78 and comprising SEQ ID NO: 224; For example, a tracr consisting of; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 78 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0019] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 79; (b) SEQ ID NO: 80; or (c) SEQ ID NO: 81 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0020] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 82 and comprising SEQ ID NO: 224; For example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 82 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, it consists of tracr; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 83 and comprising SEQ ID NO: 224; For example, a tracr consisting of; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 83 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0021] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 84; (b) SEQ ID NO: 85; or (c) SEQ ID NO: 86 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0022] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 87 and comprising SEQ ID NO: 224; For example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 87 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, it consists of tracr; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 88 and comprising SEQ ID NO: 224; For example, a tracr consisting of; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 88 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0023] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 89; (b) SEQ ID NO: 90; or (c) SEQ ID NO: 91 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0024] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 92 and comprising SEQ ID NO: 224; For example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 92 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, it consists of tracr; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 93 and comprising SEQ ID NO: 224; For example, a tracr consisting of; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 93 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0025] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 94; (b) SEQ ID NO: 95; or (c) SEQ ID NO: 96 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0026] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 97 and comprising SEQ ID NO: 224; For example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 97 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, it consists of tracr; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 98 and comprising SEQ ID NO: 224; For example, a tracr consisting of; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 98 and a crRNA comprising, e.g., SEQ ID NO: 73 For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0027] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 99; (b) SEQ ID NO: 100; or (c) SEQ ID NO: 101 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0028] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 102 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 102 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 103 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 103 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0029] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 104; (b) SEQ ID NO: 105; or (c) SEQ ID NO: 106 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0030] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 107 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 107 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 108 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 108 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0031] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 109; (b) SEQ ID NO: 110; or (c) SEQ ID NO: 111 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0032] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 112 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 112 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 113 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) crRNA comprising, e.g., consisting of, SEQ ID NO: 113 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0033] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 114; (b) SEQ ID NO: 115; or (c) SEQ ID NO: 116 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0034] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 117 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 117 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 118 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 118 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0035] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 119; (b) SEQ ID NO: 120; or (c) SEQ ID NO: 121 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0036] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 122 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 122 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 123 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) crRNA comprising, e.g., consisting of, SEQ ID NO: 123 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0037] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 124; (b) SEQ ID NO: 125; or (c) SEQ ID NO: 126 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0038] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 127 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 127 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 128 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 128 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0039] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 129; (b) SEQ ID NO: 130; or (c) SEQ ID NO: 131 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0040] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 132 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 132 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 133 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 133 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0041] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 134; (b) SEQ ID NO: 135; or (c) SEQ ID NO: 136 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0042] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 137 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 137 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 138 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 138 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0043] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 139; (b) SEQ ID NO: 140; or (c) SEQ ID NO: 141 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0044] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 142 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 142 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 143 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 143 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0045] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 144; (b) SEQ ID NO: 145; or (c) SEQ ID NO: 146 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0046] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 147 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 147 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 148 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 148 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0047] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 149; (b) SEQ ID NO: 150; or (c) SEQ ID NO: 151 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0048] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 152 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 152 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 153 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 153 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0049] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 154; (b) SEQ ID NO: 155; or (c) SEQ ID NO: 156 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0050] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 157 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 157 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 158 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 158 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0051] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 159; (b) SEQ ID NO: 160; or (c) SEQ ID NO: 161 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0052] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 162 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 162 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 163 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 163 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0053] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 164; (b) SEQ ID NO: 165; or (c) SEQ ID NO: 166 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0054] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 167 and a crRNA comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 167 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 168 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 168 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0055] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 169; (b) SEQ ID NO: 170; or (c) SEQ ID NO: 171 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0056] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 172 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 172 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 173 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) crRNA comprising, e.g., consisting of, SEQ ID NO: 173 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0057] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ ID NO: 176 The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0058] In one embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence: (a) a crRNA comprising, e.g., consisting of, SEQ ID NO: 177 and comprising SEQ ID NO: 224 , for example, tracr consisting of it; (b) a crRNA comprising, e.g., consisting of, SEQ ID NO: 177 and comprising SEQ ID NO: 73; For example, tracr consisting of it; (c) a crRNA comprising, e.g., consisting of, SEQ ID NO: 178 and comprising SEQ ID NO: 224 , for example consisting of tracr; or (d) a crRNA comprising, e.g., consisting of, SEQ ID NO: 178 and comprising SEQ ID NO: 73; For example, tracr The present invention provides a gRNA molecule comprising, e.g., consisting of:

[0059] In one aspect, including in any of the above aspects and embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: A gRNA molecule is provided, wherein: a) a CRISPR system (e.g., as described herein) comprising a gRNA molecule; When the gRNA molecule (RNP) is introduced into cells, the indels are inserted into the targeting domain of the gRNA molecule. formed at or near the target sequence; and / or b) a CRISPR system comprising a gRNA molecule (e.g., as described herein) RNP) into a cell, a deletion including, for example, substantially all of the sequence is Complementary to the gRNA targeting domain in the HBG1 promoter region (e.g., gRNA At least 90% complementary to the targeting domain, e.g., perfectly complementary to the gRNA targeting domain and a sequence complementary to the gRNA targeting domain in the HBG2 promoter region (e.g., For example, at least 90% complementary to the gRNA targeting domain, e.g., at least 90% complementary to the gRNA targeting domain. In an embodiment, an indel is formed between a non-deleted sequence and a sequence that is completely complementary to the deleted sequence. It does not contain nucleotides from HPFH or transcription factor binding sites.

[0060] In one aspect, including in any of the above aspects and embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: gRNA molecules, wherein a CRISPR system (e.g., the present invention) comprising the gRNA molecules is provided. When an RNP (as described herein) is introduced into a population of cells, indels are generated in the population. At least about 15% of the cells, e.g., at least about 17%, e.g., at least about 20%, e.g., For example, at least about 30%, for example, at least about 40%, for example, at least about 50%, e.g. For example, at least about 55%, for example, at least about 60%, for example, at least about 70%, for example to a target sequence that is at least about 75% complementary to the targeting domain of the gRNA molecule, or In some aspects, including those in any of the above aspects and embodiments, In the present invention, the indel is a sequence of at least one nucleotide in the HBG1 promoter region or In an embodiment, the HBG2 promoter region comprises at least one nucleotide. At least about 15% of the cells in the population contain at least one nucleotide sequence in the HBG1 promoter region. Indels containing nucleotides and at least one nucleotide in the HBG2 promoter region Certain aspects, including those in any of the foregoing aspects and embodiments, include indels comprising: In the present study, an indel containing at least one nucleotide in the HBG1 promoter region was identified. The percentage of cells in the population that contain at least about 5%, e.g., at least about 10%, e.g., For example, at least about 20%, e.g., at least about 30%, of the HBG2 promoter region. The percentage of cells in the population that contain indels containing at least one nucleotide differs In embodiments, indels are measured by next generation sequencing (NGS). do.

[0061] In one aspect, including in any of the above aspects and embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: gRNA molecules, wherein a CRISPR system (e.g., the present invention) comprising the gRNA molecules is provided. When the RNP (as described herein) is introduced into cells, the expression of fetal hemoglobin is , in said cell or its progeny, e.g., its erythroid progeny, e.g., its erythroid cell progeny In embodiments, a CRISPR system (e.g., When an RNP (as described herein) is introduced into a population of cells, the population or its offspring The percentage of F cells in a population of progeny, e.g., their erythroid progeny, e.g., their red blood cell progeny The experiment involves a population of cells that have not been transfected with gRNA molecules or their progeny, e.g., their red blood cells. a small percentage of F cells in the population of its erythroid progeny, e.g., at least about 15%, for example at least about 17%, for example at least about 20%, for example at least at least about 25%, for example at least about 30%, for example at least about 35%, for example at least In embodiments, the cells or their progeny, e.g., their erythroid lineages, increase by about 40%. The progeny, e.g., the red blood cell progeny thereof, have at least about 6 picograms per cell (e.g., At least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms, or about 9 to about 10 picograms) produces fetal hemoglobin.

[0062] In one aspect, including in any of the above aspects and embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: gRNA molecules, wherein a CRISPR system (e.g., the present invention) comprising the gRNA molecules is provided. When introduced into cells, the RNPs (as described herein) can be used for, for example, next-generation sequencing. Off-target insertion as detectable by nucleotide insertion assays and / or The del is not formed in the cell, e.g., the HBG1 and / or HBG2 promoters Off-target indications outside the target region (e.g., within the coding region of a gene) No nuclei are formed.

[0063] In one aspect, including in any of the above aspects and embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: gRNA molecules, wherein a CRISPR system (e.g., the present invention) comprising the gRNA molecules is provided. When introduced into a population of cells, RNPs (as described herein) can be used to generate, for example, next generation sequencing. Off-target cleavage as detectable by sequencing and / or nucleotide insertion assays Twin dels, e.g., outside the HBG1 and / or HBG2 promoter regions (e.g., Off-target indels in a gene (e.g., within the coding region of a gene) occur in approximately 100% of cells in a population of cells. detected in more than 5%, such as more than about 1%, such as more than about 0.1%, such as more than about 0.01% do not have.

[0064] In certain aspects, including in any of the foregoing aspects and embodiments, the cells are mammalian cells. The cell population may be (or may comprise) a mammalian cell, a primate cell, or a human cell, e.g., a human The cells are (or the population of cells comprises) HSPCs, e.g., For example, HSPCs are CD34+, e.g., HSPCs are CD34+CD90+ In embodiments, the cells are autologous to the patient receiving the cells. In embodiments, the cells are allogeneic to the patient to whom they are administered.

[0065] In some embodiments, the gRNA molecules described herein, genome editing systems (e.g., , CRISPR system) and / or method may comprise or have one or more of the following characteristics: For example, in a cell as described herein, which results in: (a) at least about 40%, at least about 50%, of the cells of a population of cells described herein %, at least about 60%, at least about 70%, at least about 80%, at least about 90 %, at least about 95%, at least about 96%, at least about 97%, at least about 98% % or at least about 99% correspond to the targeting domain of the gRNA molecules described herein. containing indels in or near the complementary genomic DNA sequence, and optionally the indels are 2 to 7, and optionally, the cells of the population are selected from the indels shown in 2 to 5,249,833, not including the deletion of nucleotides located between the -strands (hg38) ; (b) The cells (e.g., populations of cells) described herein are differentiated cells of the erythroid lineage (e.g., The differentiated cells have the ability to differentiate into, for example, non-modified cells (e.g., red blood cells), and the differentiated cells can be differentiated into, for example, non-modified cells (e.g., exhibiting increased levels of fetal hemoglobin relative to the population of cells; (c) The populations of cells described herein are populations of differentiated cells, e.g., cells of the erythroid lineage. The differentiated cell population can be, for example, an increased proportion of F cells relative to a population of unmodified cells (e.g., at least about 15% of F cells) , at least about 20%, at least about 25%, at least about 30%, or at least about 4 0% higher rate); (d) The cells (e.g., populations of cells) described herein are differentiated cells, e.g., erythroid cells. and the differentiated cells (e.g., differentiated cells) have the ability to differentiate into the differentiated cells (e.g., erythroid cells). population) is at least about 6 picograms (e.g., at least about 7 picograms) per cell , at least about 8 picograms, at least about 9 picograms, at least about 10 picograms , or about 8 to about 9 picograms, or about 9 to about 10 picograms) of fetal hemoglobin death; (e) Detected, for example, by next-generation sequencing and / or nucleotide insertion assays Possible off-target indels are formed in the cells described herein. For example, outside the HBG1 and / or HBG2 promoter region (e.g., genes, e.g., Off-target indels (e.g., within the coding region of a gene) are not formed; (f) Detected, for example, by next-generation sequencing and / or nucleotide insertion assays Possible off-target indels, e.g., in the HBG1 and / or HBG2 promoters Off-target indications outside the target region (e.g., within the coding region of a gene) A cell may comprise more than about 5% of the cells in a population of cells described herein, e.g., more than about 1%, e.g., not detectable at more than about 0.1%, e.g., more than about 0.01%; (g) The cells described herein or their progeny may optionally be a cell of any one of claims 1 to 22. or a genomic DNA sequence complementary to the targeting domain of the gRNA molecule of any one of claims 1 to 4. >16 weeks and >20 weeks post-transplant when detected by detecting nearby indels or detectable in the recipient, e.g., detectable in bone marrow, for more than 24 weeks; Detectable in peripheral blood, and optionally, the indel is selected from the indels shown in Tables 2-7. selected, and optionally the indel is a large deletion indel; (h) The populations of cells described herein are populations of differentiated cells, e.g., cells of the erythroid lineage. The differentiated cell population can be, for example, a reduced proportion of sickle cells relative to a population of unmodified cells (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least About 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% lower); and / or (i) The cells or populations of cells described herein are populations of differentiated cells, e.g., erythroid lineages. and the differentiated cell population is capable of differentiating into a population of cells (e.g., a population of red blood cells), For example, a reduced level (e.g., at least about 15%, at least at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least at least about 50%, at least about 60%, at least about 70%, at least about 80%, or The cells contain cells that produce sickle hemoglobin (HbS) at levels (at least about 90% lower).

[0066] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: 1) one or more gRNA molecules (including a first gRNA molecule) described herein, e.g., For example, one or more gRNA molecules of any of the aforementioned gRNA aspects and embodiments, and a Cas9 molecule as described herein; 2) one or more gRNA molecules (including a first gRNA molecule) described herein, e.g., For example, one or more gRNA molecules of any of the aforementioned gRNA aspects and embodiments, and a nucleic acid encoding a Cas9 molecule as described herein; 3) one or more gRNA molecules (including a first gRNA molecule) described herein, e.g., For example, encoding one or more gRNA molecules of any of the aforementioned gRNA aspects and embodiments. a nucleic acid and, for example, a Cas9 molecule as described herein; 4) One or more gRNA molecules (including the first gRNA molecule) described herein, e.g., For example, encoding one or more gRNA molecules of any of the aforementioned gRNA aspects and embodiments. a nucleic acid encoding a Cas9 molecule, e.g., as described herein; or 5) any one of 1) to 4) above and a template nucleic acid; or 6) A nucleic acid containing a sequence encoding any one of the above 1) to 4) and a template nucleic acid. A composition comprising:

[0067] In one embodiment, the present invention provides a first gRNA molecule described herein, such as the and a first gRNA molecule of any of the gRNA aspects and embodiments described herein, e.g. and a composition further comprising a Cas9 molecule as described in or inactivated S. pyogenes Cas9, e.g., Cas9 The molecule comprises SEQ ID NO: 205. In embodiments, the Cas9 molecule comprises (a) SEQ ID NO: 233 (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e) SEQ ID NO: No. 237; (f) SEQ ID NO: 238; (g) SEQ ID NO: 239; (h) SEQ ID NO: 240; (i (j) SEQ ID NO: 242; (k) SEQ ID NO: 243 or (l) SEQ ID NO: 2 44, including, for example, consisting of.

[0068] In certain aspects, including in any of the foregoing composition aspects and embodiments, The gRNA molecule and the Cas9 molecule are present in a ribonucleoprotein complex (RNP). .

[0069] In certain aspects, including in any of the foregoing composition aspects and embodiments, The invention also provides a second gRNA molecule; a second gRNA molecule and a third gRNA molecule; or a second a gRNA molecule, optionally a third gRNA molecule, and optionally a fourth gRNA molecule. wherein the second gRNA molecule, an optional third gRNA molecule, and and the optional fourth gRNA molecule are gRNA molecules described herein, e.g.,

[0023] The gRNA molecule of any of the foregoing gRNA molecule aspects and embodiments, wherein each gRNA molecule of the composition The RNA molecules are complementary to different target sequences. In an embodiment, the first gRNA molecule a second gRNA molecule, an optional third gRNA molecule, and an optional fourth gRNA molecule Two or more of the constructs are complementary to target sequences within the same gene or region. , a first gRNA molecule, a second gRNA molecule, an optional third gRNA molecule, and an optional The fourth gRNA molecule of choice may be 6000 nucleotides or less, 5000 nucleotides or less, or 5 00 or less, 400 or less, 300 or less, 200 or less, 100 or less nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, 50 nucleotides or less, 40 nucleotides or less, 30 nucleotides complementary to the target sequence separated by 10 nucleotides or less, 20 nucleotides or less, or 10 nucleotides or less In an embodiment, a first gRNA molecule, a second gRNA molecule, an optional third gRNA molecule, Two or more of the gRNA molecule and the optional fourth gRNA molecule are at least one gRNA molecule comprising a targeting domain complementary to a target sequence in the HB region; At least one gR containing a targeting domain complementary to a target sequence in the G2 promoter region In some embodiments, including in any of the foregoing composition aspects and embodiments, In some embodiments, the composition includes a first gRNA molecule and a second gRNA molecule (e.g., wherein the first gRNA molecule and the second gRNA molecule comprise: (a) a first gRNA molecule, e.g., is independently selected from and targets a non-deleted HPFH region described herein, and (b) are independently selected from the gRNA molecules of Table 1, and are complementary to different target sequences; c) are independently selected from the gRNA molecules of Table 2; and (d) are complementary to different target sequences; or (d) are independently selected from the gRNA molecules of Table 3a. and complementary to different target sequences; and (e) independently selected gRNA molecules from Table 3b. and are complementary to different target sequences; or (f) any of the preceding aspects and embodiments. The gRNA molecules are independently selected and complementary to different target sequences.

[0070] In certain aspects, including in any of the foregoing composition aspects and embodiments, the composition comprises a first gRNA molecule and a second gRNA molecule; a) The first gRNA molecule comprises: i)Chr11:5,249,833~Chr11:5,250,237(hg38); ii)Chr11:5,250,094~5,250,237(hg38); iii) Chr11:5,249,833 to 5,249,927 (hg38); or iv)Chr11:5,250,139~5,250,237(hg38) At least one nucleotide (e.g., 20 consecutive nucleotides) is complementary to a target sequence comprising: b) a second gRNA molecule comprising: i)Chr11:5,254,738~Chr11:5,255,164(hg38); ii) Chr11:5,255,022 to 5,255,164 (hg38); or iii)Chr11:5,254,738~5,254,851(hg38) At least one nucleotide (e.g., 20 consecutive nucleotides) is complementary to a target sequence comprising:

[0071] In one embodiment, with respect to the gRNA molecule component of the composition, the composition comprises a first gRNA molecule and a second gRNA molecule.

[0072] In certain aspects, including in any of the foregoing composition aspects and embodiments, Each of the gRNA molecules may be used in conjunction with a ribonucleoprotein, e.g., a Cas9 molecule, as described herein. It is found in the protein complex (RNP).

[0073] In certain aspects, including in any of the foregoing composition aspects and embodiments, The composition includes a template nucleic acid, the template nucleic acid being located at or near a target sequence of a first gRNA molecule. In an embodiment, the template nucleic acid comprises a nucleotide corresponding to a nucleotide in ( a) human β-globin, e.g., containing one or more of the mutations G16D, E22A, and T87Q (b) encoding human β-globin or a fragment thereof; or (b) encoding human γ-globin or a fragment thereof It includes nucleic acids that

[0074] In certain aspects, including in any of the foregoing composition aspects and embodiments, The composition is formulated in a medium suitable for electroporation.

[0075] In certain aspects, including in any of the foregoing composition aspects and embodiments, Each of the gRNA molecules of the composition may be combined with a Cas9 molecule as described herein to form an RNP. wherein each of said RNPs is less than about 10 uM, such as less than about 3 uM, for example less than about 1 uM less than about 0.5 uM, for example less than about 0.3 uM, for example less than about 0.1 uM In an embodiment, the RNP is at a concentration of about 1 uM. The NPs are at a concentration of about 2 uM. In embodiments, the concentration is, for example, as described herein. The concentration of RNP in a composition comprising cells as described above, optionally including cells and RNP. Compositions containing P are suitable for electroporation.

[0076] In one embodiment, the present invention provides one or more gRNA molecules described herein, e.g., A gene encoding one or more gRNA molecules of any of the foregoing gRNA molecule aspects and embodiments. In embodiments, the nucleic acid encodes one or more gRNA molecules. The promoter may be operably linked to a sequence that encodes an RNA promoters recognized by polymerase II or RNA polymerase III Or, for example, the promoter is a U6 promoter or a HI promoter.

[0077] In certain aspects, including any of the foregoing nucleic acid aspects and embodiments, the nucleic acid The acid can be a Cas9 molecule, e.g., SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: No. 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO: 244 In embodiments, the Cas9 molecule further encodes a Cas9 molecule comprising, e.g., consisting of, The nucleic acid may comprise a promoter, e.g., a promoter operably linked to a sequence encoding a Cas9 molecule. For example, the EF-1 promoter, the CMV IE gene promoter, the EF-1α promoter, Contains the ubiquitin C promoter or the phosphoglycerate kinase (PGK) promoter nothing.

[0078] In one aspect, the invention includes a nucleic acid of any of the foregoing nucleic acid aspects and embodiments. In embodiments, the vector is a lentiviral vector, an adenovirus vector, or a recombinant vector. Noviral vectors, adeno-associated virus (AAV) vectors, herpes simplex virus ( HSV) vectors, plasmids, minicircles, nanoplasmids and RNA vectors is selected from the group consisting of:

[0079] In one aspect, the present invention provides a method for identifying a cell (e.g., a population of cells) by targeting a target sequence within said cell. or near it (e.g., modifying the structure (e.g., sequence) of a nucleic acid). and subjecting the cells (e.g., population of cells) to: 1) one or more gRNA molecules described herein (e.g., embodiments of the gRNA molecules described above) and one or more gRNA molecules of any of the embodiments) and, e.g., C as described herein. as9 molecule; 2) one or more gRNA molecules described herein (e.g., embodiments of the gRNA molecules described above) and one or more gRNA molecules of any of the embodiments) and, e.g., C as described herein. a nucleic acid encoding an as9 molecule; 3) one or more gRNA molecules described herein (e.g., embodiments of the gRNA molecules described above) and one or more gRNA molecules of any of the embodiments) and Cas9 molecules as described in the book; 4) one or more gRNA molecules described herein (e.g., embodiments of the gRNA molecules described above) and one or more gRNA molecules of any of the embodiments) and a nucleic acid encoding a Cas9 molecule described herein; 5) any one of 1) to 4) above and a template nucleic acid; 6) A nucleic acid comprising a sequence encoding any one of 1) to 4) above and a template nucleic acid; 7) A composition as described herein, e.g., any of the aspects and embodiments of the composition described above. a composition; or 8) A vector as described herein, e.g., any of the aspects and embodiments of the vectors described above. That vector In one embodiment, a method is provided comprising contacting (e.g., introducing) a

[0080] In certain aspects, including in any of the aforementioned method aspects and embodiments, gR A nucleic acid encoding a NA molecule or a gRNA molecule and a Cas9 molecule or a nucleic acid encoding a Cas9 molecule. In another embodiment, the gRNA molecule or the gR nucleic acid is formulated into a single composition. The nucleic acid encoding the NA molecule and the Cas9 molecule or the nucleic acid encoding the Cas9 molecule are In some embodiments, the two or more compositions are formulated simultaneously or sequentially. It will then be delivered.

[0081] The methods described herein, including those in any of the foregoing method aspects and embodiments, In some embodiments of the method, the cell is an animal cell, e.g., the cell is a mammalian cell, a primate cell, cells or human cells, for example, the cells are hematopoietic stem and progenitor cells (HSPCs) (e.g., , a population of HSPCs), e.g., the cells are CD34+ cells, e.g., the cells are In an embodiment of the methods described herein, the cells is disposed in a composition comprising a population of cells enriched for CD34+ cells. In embodiments of the methods described herein, the cells (e.g., populations of cells) are derived from bone marrow, The cells are isolated from mobilized peripheral blood or umbilical cord blood. Thus, the cells may be autologous or allogeneic to the patient receiving the cells, e.g., autologous. This is the origin.

[0082] The methods described herein, including those in any of the foregoing method aspects and embodiments, In certain embodiments of the method, a) the modification comprises modifying a targeting domain of one or more gRNA molecules complementary to the targeting domain of the one or more gRNA molecules. or b) the modification results in an indel in or near a non-targeted genomic DNA sequence; or complementary in the promoter region to the targeting domain of one or more gRNA molecules (e.g., g At least 90% complementary to the RNA targeting domain, e.g., perfectly complementary to the gRNA targeting domain (complementary to HBG2) and target one or more gRNA molecules in the HBG2 promoter region. complementary to the gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain, e.g., between the sequence containing the gRNA targeting domain and the sequence containing the gRNA targeting domain (e.g., fully complementary to the gRNA targeting domain), e.g., In an embodiment of this method, the indel is about 40 less than nucleotides, for example less than 30 nucleotides, for example less than 20 nucleotides, for example The insertion or deletion is less than 10 nucleotides, for example a single nucleotide deletion.

[0083] The methods described herein, including those in any of the foregoing method aspects and embodiments, In some embodiments of the method, the method comprises: For example, at least about 17%, for example, at least about 20%, for example, at least about 30%, e.g. For example, at least about 40%, for example, at least about 50%, for example, at least about 55%, for example At least about 60%, such as at least about 70%, such as at least about 75% modified This results in a population of cells that contain indels, e.g., indels.

[0084] The methods described herein, including those in any of the foregoing method aspects and embodiments, In some embodiments of the method, the modification induces differentiation into differentiated cells of the erythroid lineage (e.g., red blood cells). The differentiated cells may be, for example, non-modified cells. A cell (e.g., a population of cells) exhibits increased levels of fetal hemoglobin.

[0085] The methods described herein, including those in any of the foregoing method aspects and embodiments, In some embodiments of the method, the modification is performed on a population of differentiated cells, such as a population of cells of the erythroid lineage (e.g., For example, a population of red blood cells) and For example, an increased proportion of F cells relative to a population of unmodified cells (e.g., fewer F cells) At least about 15%, at least about 20%, at least about 25%, at least about 30%, or less at least about 40% higher).

[0086] The methods described herein, including those in any of the foregoing method aspects and embodiments, In some embodiments of the method, the modification is in a differentiated cell, e.g., a cell of the erythroid lineage (e.g., an erythroid cell). and the differentiated cells are at least about 6 per cell. picograms (e.g., at least about 7 picograms, at least about 8 picograms, at least at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms, or about 9 ~ about 10 picograms of fetal hemoglobin.

[0087] In some aspects, the present invention relates to the methods described herein, including the aforementioned method aspects and and a cell modified by the method of any of the embodiments.

[0088] In some aspects, the present invention relates to the methods described herein, including the aforementioned method aspects and and a cell obtainable by the method of any of the embodiments.

[0089] In one embodiment, the present invention provides a first gRNA molecule described herein, such as the The first gRNA molecule of any of the aspects or embodiments of the gRNA molecule described herein The compositions of any of the aspects or embodiments of the compositions described herein, for example, A nucleic acid as described, e.g., a nucleic acid of any of the aspects or embodiments of the nucleic acid described above, or a vector as described in any of the aspects or embodiments of the vectors described above. The present invention provides a cell comprising:

[0090] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments of the cells, the cells are, e.g., transfected with a Cas9 molecule described herein, e.g., SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, A Cas9 molecule comprising any one of SEQ ID NO: 242, SEQ ID NO: 243, or SEQ ID NO: 244. Further includes:

[0091] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments of the cells, the cells are transfected with a second gRNA molecule described herein, e.g., a pre- The second gRNA molecule or the gRNA of any of the aspects or embodiments of the gRNA molecule described above. contains, has contained, or will contain, nucleic acid encoding a molecule, The first gRNA molecule and the second gRNA molecule contain non-identical targeting domains.

[0092] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments of the cells, expression of fetal hemoglobin is modified to include a gRNA molecule. the cell or its progeny (e.g., its red blood cells) to cells or their progeny of the same cell type that are not It is increased in blood cell progeny, such as its red blood cell progeny.

[0093] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments of the cells, the cells are differentiated cells, e.g., cells of the erythroid lineage (e.g., erythroid cells). The differentiated cells have the ability to differentiate into cells, for example, modified to contain a gRNA molecule. They exhibit increased levels of fetal hemoglobin relative to the same cell type.

[0094] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments of the invention, differentiated cells (e.g., cells of the erythroid lineage, e.g., red blood cells) are For example, a small number of differentiated cells of the same type that have not been modified to contain the gRNA molecule at least about 6 picograms (e.g., at least about 7 picograms, at least about 8 picograms) , at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms , or about 9 to about 10 picograms) of fetal hemoglobin.

[0095] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments, the cells are treated with a stem cell proliferation agent, such as a) (1r,4r)-N 1 -( 2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[ 4,5-b]indol-4-yl)cyclohexane-1,4-diamine; b) methyl 4 -(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indo c) 4-(2-(2-(benzo[b]thiophene-3-yl)-7-carboxylate; d) (yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-phenylindol-3-yl)ethylamino)- (fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; or e ) combinations of these (e.g., (1r,4r)-N 1 -(2-benzyl-7-(2-methyl (2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indole-4 -yl)cyclohexane-1,4-diamine and (S)-2-(6-(2-(lH-yne) (5-fluoropyridin-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H- a stem cell proliferation agent selected from the group consisting of (a) a combination of (purin-9-yl)propan-l-ol; In an embodiment, the stem cells are contacted with the The cell proliferation agent is (S)-2-(6-(2-(lH-indol-3-yl)ethylamino) -2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propane-l- It's an oar.

[0096] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments of the cell, the cell is a) transfected with a gRNA molecule described herein, e.g., a gRNA molecule described above Complementary to the targeting domain of the gRNA molecule of any of the aspects or embodiments of the gRNA molecule or b) an indel in or near a target genomic DNA sequence; or Embodiments of the gRNA molecules described herein, such as the gRNA molecules described above, in the target region or complementary to the targeting domain of the gRNA molecule of any of the embodiments (e.g., gRNA target At least 90% complementary to the targeting domain, e.g., fully complementary to the gRNA targeting domain ) and a gRNA molecule described herein in the HBG2 promoter region, e.g. For example, the targeting domain of a gRNA molecule of any of the foregoing gRNA molecule aspects or embodiments. complementary to (e.g., at least 90% complementary to the gRNA targeting domain, e.g., gRNA a sequence between the targeting domain (fully complementary to the targeting domain), e.g., substantially the entire targeting domain, In some embodiments, an indel is a deletion of less than about 40 nucleotides, For example, less than 30 nucleotides, for example, less than 20 nucleotides, for example, less than 10 nucleotides An indel is a single nucleotide deletion.

[0097] The cells described herein, including those in any of the foregoing cell aspects and embodiments, In some embodiments, the cell is an animal cell, e.g., the cell is a mammalian cell, a primate cell, or the like. In one embodiment, the cells are hematopoietic stem and progenitor cells (HSPCs). ) (e.g., a population of HSPCs), e.g., the cells are CD34+ cells, e.g., In some embodiments, the cells are CD34+CD90+ cells. The cell population is isolated from bone marrow, mobilized peripheral blood, or umbilical cord blood. In an embodiment, the cells are autologous to the patient to whom the cells are administered. The cells are of allogeneic origin to the patient to whom they are administered.

[0098] In one aspect, the present invention provides a population of cells described herein, e.g., a population of cells described herein. a population of cells comprising the cells of any of the preceding cell aspects and embodiments. In an aspect, the present invention provides a population of cells, wherein the cells of the population of cells at least about 50%, such as at least about 60%, such as at least about 70%, At least about 80%, e.g., at least about 90% (e.g., at least about 95%, At least about 96%, at least about 97%, at least about 98%, or at least about 99% a cell as described herein, such as a cell of any of the foregoing cell aspects and embodiments. In some embodiments, the population of cells (e.g., cells of the population of cells) is a population of differentiated cells, e.g., For example, the cells have the ability to differentiate into a population of cells of the erythroid lineage (for example, a population of red blood cells), A population of cells, e.g., an increase in F cells relative to a population of unmodified cells of the same type a proportion (e.g., at least about 15%, at least about 17%, at least about 2% of F cells) 0%, at least about 25%, at least about 30%, or at least about 40% higher In embodiments, the population of differentiated cells has an average of at least about 6 F cells per cell. picograms (e.g., at least about 7 picograms, at least about 8 picograms, at least at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms, or about 9 ~ about 10 picograms of fetal hemoglobin.

[0099] In certain aspects, including in any of the aspects and embodiments of the populations of cells described above The present invention provides: 1) at least 1e6 CD34+ cells / kg of body weight of the patient to whom the cells are administered; 2) at least 2e6 CD34+ cells / kg of patient's body weight to be administered; 3) fewer 4) at least 4 e 6) CD34+ cells / kg of patient's body weight to be administered; or 5) 2e6 to 10e6 C The population of cells comprises D34+ cells / kg body weight of the patient to whom the cells are to be administered. In the present invention, at least about 40%, e.g., at least about 50%, (e.g., at least about 10%) of the population of cells at least about 60%, at least about 70%, at least about 80%, or at least about 90% ) are CD34+ cells. In embodiments, at least about 5% of the cells in this population, e.g., For example, at least about 10%, for example, at least about 15%, for example, at least about 20%, for example In some embodiments, at least about 30% of the cells are CD34+CD90+ cells. The population may be derived from cord blood, peripheral blood (e.g., mobilized peripheral blood), or bone marrow, e.g., bone marrow-derived. In embodiments, the population of cells comprises mammalian cells, e.g., human cells, e.g., In embodiments, the population of cells is autologous to the patient to whom it is administered. In other embodiments, the population of cells is derived from the same organism as the patient to whom it is administered. It is of interspecific origin.

[0100] In some embodiments, the present invention relates to the cells described herein, such as the aforementioned cell embodiments and and any of the cells or populations of cells described herein, such as any of the aforementioned cells. Compositions are provided that include the population of cells of any of the population aspects and embodiments. The composition may be stored in a pharmaceutically acceptable medium, such as a pharmaceutically acceptable carrier suitable for cryopreservation. This includes media such as:

[0101] In some embodiments, the present invention relates to the cells described herein, such as the aforementioned cell embodiments and and any of the cells of the embodiments, populations of cells described herein, e.g., populations of the aforementioned cells. A population of cells of any of the group aspects and embodiments or compositions described herein, e.g. administering to a patient a composition according to any of the preceding composition aspects and embodiments; A method for treating hemoglobinopathies is provided.

[0102] In some embodiments, the present invention relates to the cells described herein, such as the aforementioned cell embodiments and and any of the cells of the embodiments, populations of cells described herein, e.g., populations of the aforementioned cells. A population of cells of any of the group aspects and embodiments or compositions described herein, e.g. administering to a patient a composition according to any of the preceding composition aspects and embodiments; A method for increasing fetal hemoglobin expression in a mammal is provided. The hemoglobinopathy is β-thalassemia. In embodiments, the hemoglobinopathy is sickle cell disease. It is a red blood cell disease.

[0103] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) A cell (e.g., a population of cells) (e.g., HSPCs) Steps to provide; (b) culturing the cells (e.g., the population of cells) in a cell culture medium containing a stem cell proliferation agent. ex vivo culturing of the (c) e.g., a first gRNA molecule described herein, e.g., a gRNA molecule described above The first gRNA molecule of any of the aspects and embodiments; a nucleic acid encoding the first gRNA molecule an acid molecule; any of the compositions described herein, including any of the aspects and embodiments of the aforementioned compositions; or any of the compositions; or any of the vectors described herein, such as any of the foregoing aspects and embodiments. a method for preparing cells (e.g., a population of cells) comprising introducing into said cells a vector In an embodiment of this method, after the introduction of step (c), the cells (e.g., a population of cells) can be differentiated cells (e.g., a population of differentiated cells), e.g., of the erythroid lineage. Cells (e.g., a population of cells of the erythroid lineage), e.g., red blood cells (e.g., a population of red blood cells) The differentiated cells (for example, a population of differentiated cells) can be differentiated, for example, by the steps (c) produces increased fetal hemoglobin relative to the same cells not subjected to (c). In one embodiment, the stem cell proliferation agent is a) (1r,4r)-N1-(2-benzyl-7-( 2-Methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indo b) methyl 4-(3-piperidine- 1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxy c) 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl d) (S)-2-(6-(2-[(2-amino-9H-purin-6-ylamino)ethyl]phenol; -(lH-indol-3-yl)ethylamino)-2-(5-fluoropyridine-3- e) a combination thereof; or f) 9H-purin-9-yl)propan-l-ol; (e.g., (1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazolium) (4,5-b)indol-5-yl-9H-pyrimido[4,5-b]indol-4-yl)cyclohexa Indole-1,4-diamine and (S)-2-(6-(2-(lH-indol-3-yl)ethoxy)-2-methyl-2-propanediol (ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)purine In an embodiment, the stem cell proliferation agent is (S )-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-(5-fluoro The compound is (9H-pyridin-3-yl)-9H-purin-9-yl)propan-1-ol. In this study, the cell culture medium contained thrombopoietin (Tpo), Flt3 ligand (Flt In some embodiments, the cell culture medium comprises human stem cell factor (SCF). In embodiments, the cell culture medium further comprises interleukin-6 (IL-6). Myopoietin (Tpo), Flt3 ligand (Flt-3L) and human stem cell factor (SCF ) at a concentration ranging from about 10 ng / mL to about 1000 ng / mL, for example, In an embodiment, the cells are each at a concentration of 50 ng / mL, for example, 50 ng / mL each. The cell culture medium contains human interleukin-6 (IL-6) at approximately 10 ng / mL to approximately 1000 Concentrations in the ng / mL range, e.g., concentrations of about 50 ng / mL, e.g., concentrations of 50 ng / mL In some embodiments, the cell culture medium contains a stem cell proliferation agent at a concentration ranging from about 1 nM to about 1 mM. For example, a concentration in the range of about 1 uM to about 100 nM, for example, about 500 nM to about 750 nM In embodiments, the cell culture medium contains a stem cell proliferation agent at a concentration of about 500 nM. In embodiments, the cell culture medium contains a stem cell proliferation agent at a concentration of about 700 nM, for example, 500 nM. Included at a concentration of 50 nM, for example at a concentration of 750 nM.

[0104] In embodiments of the method of preparing cells (e.g., a population of cells), the culturing of step (b) comprises , including a culture period before the introduction of step (c), for example, a culture period before the introduction of step (c) The period is at least 12 hours, for example, from about 1 day to about 12 days, for example, about 1 day For example, a period of about 1 to about 6 days, for example, a period of about 1 to about 3 days, for example, a period of about 1 to about 2 days For example, for a period of about 2 days. In an embodiment of a method for preparing cells (e.g., a population of cells), including those comprising the steps of: The culturing in b) includes the culturing period after the introduction of step (c), for example, The culture period after the introduction is at least 12 hours, for example, about 1 day to about 12 days. For example, a period of about 1 to about 6 days, for example, a period of about 2 to about 4 days, for example, about 2 days Alternatively, the period is about 3 days, or about 4 days. and methods of preparing cells (e.g., populations of cells), including those in any of the embodiments. In this embodiment, the population of cells is, for example, cells that have not been cultured by step (b). at least four-fold, such as at least five-fold, such as at least ten-fold do.

[0105] In any of the aspects and embodiments of the aforementioned methods, cells (e.g., cells In an embodiment of the method for preparing a population of HIV-1 strains, the introducing step (c) is performed by electroporation. In one embodiment, electroporation is performed with 1 to 5 pulses, for example, 1 pulse. Each pulse has a pulse voltage in the range of 700 volts to 2000 volts, and In one embodiment, the electroporation In some embodiments, a pulse (or two or more pulses) may comprise, e.g., consist of, one pulse. The power supply voltage is in the range of 1500 to 1900 volts, for example 1700 volts. In an embodiment, the pulse duration of one or more pulses is between 10 ms and 40 ms. range, for example 20 ms.

[0106] In any of the aspects and embodiments of the aforementioned methods, cells (e.g., cells In an embodiment of the method for preparing a population of cells (e.g., cells) provided in step (a), The population of cells is a human cell (e.g., a population of human cells). In some embodiments, the method includes preparing a cell (e.g., a population of cells), including in any of the following states: In this case, the cells (e.g., population of cells) provided in step (a) may be derived from bone marrow, peripheral blood (e.g., For example, the cells are isolated from mobilized peripheral blood or umbilical cord blood. In some embodiments of the method for preparing cells (e.g., a population of cells), including those The cells (e.g., population of cells) provided in step (a) can be isolated from bone marrow, e.g., a heterologous It is isolated from the bone marrow of patients suffering from hemoglobinopathies.

[0107] In any of the aspects and embodiments of the aforementioned methods, cells (e.g., cells In an embodiment of the method for preparing a population of cells, the population of cells provided in step (a) is a CD Enriched for 34+ cells.

[0108] In any of the aspects and embodiments of the aforementioned methods, cells (e.g., cells In embodiments of the method for preparing a population of cells (e.g., a population of cells), after the introduction of step (c), The populations are cryopreserved.

[0109] In any of the aspects and embodiments of the aforementioned methods, cells (e.g., cells In embodiments of the method for preparing a population of cells (e.g., a population of cells), after the introduction of step (c), a) a sequence in a genomic DNA sequence complementary to the targeting domain of the first gRNA molecule; or b) an indel in or near the first g complementary to the targeting domain of the RNA molecule (e.g., at least 9 nucleotides long, complementary to the gRNA targeting domain) 0% complementary, e.g., fully complementary to the gRNA targeting domain) and the HBG2 promoter. complementary in the targeting domain of the first gRNA molecule (e.g., the gRNA targeting domain) At least 90% complementary to the targeting domain, e.g., fully complementary to the gRNA targeting domain ) and including, for example, substantially all of the sequence.

[0110] In any of the aspects and embodiments of the aforementioned methods, cells (e.g., cells In an embodiment of the method for preparing a population of cells, after the introduction of step (c), at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the first gRNA molecule The targeting domain of the genomic DNA sequence of the present invention comprises an indel in or near the genomic DNA sequence complementary to the targeting domain of the genomic DNA sequence of the present invention.

[0111] In some aspects, the present invention relates to the cell preparation methods described herein, e.g., as described above. The method for preparing cells (e.g., a population of cells) according to any of the embodiments and aspects of the present invention may be used. Thus, cells (eg, populations of cells) obtainable therefrom are provided.

[0112] In one aspect, the present invention provides a method of treating a hemoglobinopathy in a human patient, comprising: a cell as described herein, such as a cell of any of the foregoing cell aspects and embodiments; or any of the populations of cells described herein, such as any of the aspects and embodiments of the populations of cells described above. The present invention provides a method for the treatment of a human patient comprising administering to the human patient a composition comprising any of the populations of cells. In an embodiment, the hemoglobinopathy is β-thalassemia. Globinopathy is a sickle cell disease.

[0113] In one aspect, the present invention provides a method for increasing fetal hemoglobin expression in a human patient. Thus, the cells described herein, such as any of the aforementioned cell aspects and embodiments, or a population of cells as described herein, such as aspects and embodiments of the population of cells described above. and administering to said human patient a composition comprising a population of cells of any of the above. In an embodiment, the human patient is suffering from β-thalassemia. Patients suffer from sickle cell disease.

[0114] Aspects of the Method of Treating Hemoglobinopathies or Increasing Fetal Hemoglobin Expression In the present study, the human patient is provided with at least about 1e6 cells (e.g., For example, cells described herein), e.g., at least about 1 e per kg of body weight of a human patient administered a composition comprising 6 CD34+ cells (e.g., cells described herein) Aspects of the Method of Treating Hemoglobinopathies or Increasing Fetal Hemoglobin Expression In the present study, the human patient is provided with at least about 2e6 cells (e.g., For example, the cells described herein), e.g., at least about 2 e / kg body weight of a human patient administered a composition comprising 6 CD34+ cells (e.g., cells described herein) Aspects of the Method of Treating Hemoglobinopathies or Increasing Fetal Hemoglobin Expression In this study, a human patient received approximately 2e6 cells per kg of human patient body weight (e.g., and the cells described in the document), e.g., approximately 2e6 CD34+ cells per kg of body weight in a human patient. (e.g., cells described herein). In an embodiment of the method of treating or increasing fetal hemoglobin expression, the human patient , at least about 3e6 cells per kg of body weight of a human patient (e.g., cells), e.g., at least about 3e6 CD34+ cells per kg of body weight in a human patient. (e.g., cells described herein). In an embodiment of the method of treating or increasing fetal hemoglobin expression, the human patient , approximately 3e6 cells (e.g., cells described herein) per kg of body weight in a human patient , for example, about 3e6 CD34+ cells per kg of body weight in a human patient (e.g., The method of treating hemoglobinopathy or the method of treating fetuses is also described. In an embodiment of the method for increasing fetal hemoglobin expression, the human patient is About 2e6 to about 10e6 cells (e.g., cells described herein) per g, e.g. Approximately 2e6 to approximately 10e6 CD34+ cells per kg of body weight of a human patient (e.g., The patient is administered a composition comprising the cells (as described in the document).

[0115] In one aspect, the present invention provides a method for the treatment of a gR as described herein for use as a medicament. a gRNA molecule, such as a gRNA molecule of any of the aspects and embodiments of the gRNA molecule described above; A composition as described herein, for example, any of the composition aspects and embodiments described above. , a nucleic acid as described herein, such as a nucleic acid of any of the foregoing nucleic acid aspects and embodiments; A vector as described herein, such as any of the aspects and embodiments of the vectors described above. a vector; a cell as described herein, such as any of the aspects and embodiments of the cell described above. or a population of cells as described herein, such as aspects and implementations of the population of cells. A population of cells of any morphology is provided.

[0116] In one aspect, the present invention provides a method for the manufacture of a medicament comprising administering to a subject a compound of formula (I) or (II) as described herein. an RNA molecule, such as a gRNA molecule according to any of the gRNA molecule aspects and embodiments described above; Compositions described herein, including any of the aspects and embodiments of the compositions described above. a nucleic acid as described herein, such as a nucleic acid of any of the foregoing nucleic acid aspects and embodiments a vector as described herein, such as any of the aspects and embodiments of the vectors described above; a vector of any of the aspects and embodiments of the cells described herein, such as or any of the populations of cells described herein, including embodiments and examples of the populations of cells described above. A population of cells according to any of the embodiments is provided.

[0117] In one embodiment, the present invention provides a method for treating a disease comprising administering to a subject a compound of formula (I) or (II) described herein. an RNA molecule, such as a gRNA molecule according to any of the gRNA molecule aspects and embodiments described above; Compositions described herein, including any of the aspects and embodiments of the compositions described above. a nucleic acid as described herein, such as any of the nucleic acid aspects and embodiments described above; a vector as described herein, such as any of the aspects and embodiments of the vectors described above; a vector of any of the aspects and embodiments of the cells described herein, such as or any of the populations of cells described herein, including embodiments and examples of the populations of cells described above. A population of cells according to any of the embodiments is provided.

[0118] In one embodiment, the present invention provides a method for treating a disease comprising administering to a subject a compound of formula (I) or (II) described herein. an RNA molecule, such as a gRNA molecule according to any of the gRNA molecule aspects and embodiments described above; Compositions described herein, including any of the aspects and embodiments of the compositions described above. a nucleic acid as described herein, such as any of the nucleic acid aspects and embodiments described above; a vector as described herein, such as any of the aspects and embodiments of the vectors described above; a vector of any of the aspects and embodiments of the cells described herein, such as or any of the populations of cells described herein, including embodiments and examples of the populations of cells described above. A population of cells of any of the embodiments is provided, wherein the disease is a hemoglobinopathy, e.g. Beta-thalassemia or sickle cell disease. [Brief explanation of the drawings]

[0119] [Figure 1]HbF induction 7 days after editing. For each gRNA target sequence tested, the percentage of cells with induced HbF expression, corrected for background levels based on mock transfection, is shown as the mean, with error bars indicating standard deviation. gRNA G8 against exon 2 of BCL11A served as a positive control. The dotted line at 17% indicates the threshold level selected for analysis. As indicated in the legend, various gray shades relate the degree of HbF induction to the degree of editing at the HBG1 or HBG2 target locus. [Figure 2] Editing efficiency at the HBG1 target locus. For each gRNA tested, the percentage of indels detected by NGS is shown as the average with error bars indicating the standard deviation. gRNA G8 against exon 2 of BCL11A served as a positive control. The two guides for which no NGS data was obtained are indicated by arrows. [Figure 3] Editing efficiency at the HBG2 target locus. For each gRNA tested, the percentage of indels detected by NGS is shown as the average with error bars indicating the standard deviation. gRNA G8 against exon 2 of BCL11A served as a positive control. The 16 guides for which no NGS data was available are indicated by arrows. [Figure 4] Overview of the location of high-performing gRNA target sequences (e.g., >17% HbF upregulation at day 7), known non-deletional HPFH polymorphisms and transcription factor binding sites in the HBG1 promoter region. The figure discloses SEQ ID NOs: 293-312, respectively, in order of appearance. [Figure 5] Overview of the location of high-performing gRNA target sequences (e.g., >17% HbF upregulation at day 7), known non-deletional HPFH polymorphisms and transcription factor binding sites in the HBG2 promoter region. The figure discloses SEQ ID NOs: 313-332, respectively, in order of appearance. [Figure 6]Editing efficiency at the targeted B2M locus of CD34+ HSPCs by various Cas9 variants as determined by NGS and flow cytometry. NLS = SV40 NLS; His6 (SEQ ID NO: 247) or His8 (SEQ ID NO: 248) refer to six (SEQ ID NO: 247) or eight (SEQ ID NO: 248) histidine residues, respectively; TEV = tobacco etch virus cleavage site; Cas9 = wild-type S. pyogenes Cas9 - mutants or variants are as indicated). [Figure 7] Detection and quantification of HbF+ cells by flow cytometry at 7 (black bars), 14 (light gray bars), or 21 (dark gray bars) days of erythroid differentiation after electroporation of HSPCs with RNPs containing sgRNAs of the indicated targeting domains. The percentage of HbF+ cells for control cultures not treated with sgRNA at each time point has been subtracted. Mean values ​​+ standard deviations are shown (n = 2 technical replicates). [Figure 8] Detection and quantification of HbF-positive cells by flow cytometry at 7 days (open black bars), 14 days (open light gray bars), or 21 days (open dark gray bars) of erythroid differentiation after electroporation of HSPCs with RNPs containing sgRNAs of the indicated targeting domains. The percentage of HbF+ cells for control cultures not treated with sgRNA at each time point has been subtracted. The average values ​​(bars) of two independent cell donors are shown, along with the values ​​for each donor (circles = first donor, triangles = second donor). [Figure 9]Visualization of PCR products from the indicated reactions: P1, P2, or P3, as described in the Examples, from cells after electroporation with RNPs containing sgRNAs of the indicated targeting domains or control cells not treated with sgRNA. Expected products are as follows: P1: 7.7 kb for wild-type / small indel allele or 4.9 kb inversion allele, 2.8 kb for 4.9 kb deletion allele. P2: 3.8 kb for wild-type / small indel allele, no product for 4.9 kb deletion or 4.9 kb inversion allele. P3: 1.8 kb for 4.9 kb inversion allele, no product for wild-type / small indel allele or 4.9 kb deletion allele. L = DNA reference ladder. * = DNA from this band was isolated and subjected to next-generation sequencing. [Figure 10] Schematic showing the genomic locations of primer and probe binding sites for the digital droplet PCR assay to quantify the 4.9 kb deletion. The primers (5.2 kb Fwd and 5.2 kb Rev) and probe (FAM probe) bind to an intergenic site downstream of HBG2 and upstream of HBG1. The probe has a second binding site upstream of HBG2, but that region is not bound by the primers. The regions where the targeting domains are located in the HBG1 and HBG2 promoters are indicated. If the sequence between the two targeting domain regions is deleted, the primer / probe binding site between HBG1 and HBG2 will be lost. [Figure 11] Sorting scheme for HSPC subpopulations for cell samples after electroporation with RNPs containing sgRNAs with the GCR-0067 targeting domain. Dot plots of cell fluorescence after immunostaining targeted to the indicated cell surface markers are shown. The following populations were sorted as indicated: P5 = CMP (CD34+CD45RA-CD38+), P9 = MPP (CD34+CD45RA-CD38-CD90-CD49f-), P10 = ST-HSC (CD34+CD45RA-CD38-CD90-CD49f+), and P11 = LT-HSC (CD34+CD45RA-CD38-CD90+CD49f+). Total CD34+ cells were also sorted (not shown). [Figure 12] Percent editing of sorted HSPC subpopulations after electroporation with RNPs containing sgRNAs of the GCR-0067 targeting domain. HBG1 indels and HBG2 indels represent the percentage of small insertions and deletions identified by next-generation sequencing of PCR amplicons located in or near the HBG1 or HBG2 promoter region targeting domains, respectively (note that alleles with the 4.9 kb deletion or inversion described above are not amplified). HBG1-HBG2 deletion represents the percentage of alleles with the 4.9 kb deletion, as determined by the digital droplet PCR assay described in the Examples. Total editing is an estimate calculated by adding the percentage of HBG1-HBG2 deletions to the percentage without the HBG1-HBG2 deletion and multiplying by the percentage of HBG2 indels. [Figure 13-1]Figure 13: Figure 13A shows the sum of all indels observed at the HBG1 locus in the indicated cell types after introduction of an sgRNA containing the targeting domain of GCR-0067. The indels are ordered, with the most frequently observed indel indicated at the top of each bar. The fraction of cells containing the large 4.9 kb deletion between the HBG1 and HBG2 loci is not quantified in this assay. The number within each bar of the most frequent indel indicates the number of nucleotide differences from the wild-type genomic sequence (- indicates a deletion; + indicates an insertion). Figure 13B shows the sum of all indels observed at the HBG2 locus in the indicated cell types after introduction of an sgRNA containing the targeting domain of GCR-0067. The indels are ordered, with the most frequently observed indel indicated at the top of each bar. The fraction of cells containing the large 4.9 kb deletion between the HBG1 and HBG2 loci is not quantified in this assay. The number within each bar of the most frequent indel indicates the number of nucleotide differences from the wild-type genomic sequence (- indicates a deletion; + indicates an insertion). CMP = CD34+CD45RA-CD38+ cells; MPP = CD34+CD45RA-CD38-CD90-CD49f- cells; ST-HSC = CD34+CD45RA-CD38-CD90-CD49f+ cells; and LT-HSC = CD34+CD45RA-CD38-CD90+CD49f+ cells. [Figure 13-2] (As mentioned above.) [Figure 14] Percentage of colonies corresponding to the indicated subtype, CFU-GEMM (dark gray bars), CFU-G / M / GM (medium gray bars), or BFU-E / CFU-E (light gray bars), after electroporation with RNPs containing sgRNAs of the indicated targeting domains or control cultures not treated with sgRNA. Mean values ​​+ / - standard deviations are shown (n = 2 independent donors). [Figure 15]Fold expansion of total nucleated cells (TNC), CD34-positive cells (CD34+), and CD34 and CD90 double-positive cells (CD34+CD90+) in medium containing compound 4 after electroporation with RNPs containing sgRNAs of the GCR-0067 targeting domain or control cultures not treated with sgRNA, as indicated. Mean values ​​+ / - standard deviations are shown (n = 2 independent donors). Mean values ​​(bars) of three independent cell donors are shown, along with values ​​for each donor (squares = donor A, triangles = donor B, circles = donor C). Differences between edited and control cultures were non-significant (ns) by unpaired t-test (GraphPad Prism). [Figure 16] Representative gating of cell populations by flow cytometry. Dot plots of cell fluorescence after immunostaining targeted to the indicated cell surface markers or isotype control (Isotype) are shown. Gates indicated by bold boxes were used to quantify the percentage of the indicated population and were set to exclude cells labeled with the isotype control. Only viable cells pre-gated as DAPI negative and within the cell forward and side scatter gates are shown, derived from donor C electroporated with Cas9 alone. [Figure 17] Percentage of cells with the indicated cell surface phenotype, as assessed by flow cytometry, following electroporation with RNPs containing the GCR-0067 targeting domain sgRNA (black bars) or control cultures not treated with sgRNA (gray bars). Cells were grown 2 days after electroporation of RNPs in medium containing Compound 4 and assessed by flow cytometry as described in Figure 16. Mean values ​​+ standard deviations are shown (n = 3 independent donors). There were no significant differences between edited and unedited cells for a given population by unpaired t-test (GraphPad Prism). [Figure 18-1]Figure 18: Figure 18A shows CE-MS quantification of globin subunits in mock-edited HSCs derived from a sickle cell disease patient (SCD1) that were mock-edited with Cas9 and without sgRNA. After differentiation into the erythroid lineage, the cells displayed normal levels of a-globin, absence of normal b-globin due to sickle homozygosity, high levels of sickle b-globin subunits, and low levels of fetal g-globin. Figure 18B shows CE-MS quantification of globin subunits in genome-edited HSPCs derived from a sickle cell disease patient (SCD1). After editing the HSCs, erythroid cells derived from the sample patient showed a 40% upregulation of fetal g-globin and a concomitant 50% downregulation of sickle b-globin subunits. [Figure 18-2] (As mentioned above.) [Figure 19] Schematic protocol for investigating engraftment of gene-edited cells. Transplantation of HSCs gene-edited with sgRNA containing Cas9 and the targeting domain of CR001128 (sg1128). 0.5 million human CD34+ cells were mock-edited with gRNA or gene-edited with sg1128 and then injected into 2 Gy-irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) recipients. Mice were bled at 4, 8, 12, and 16 weeks, and bone marrow cells were collected at 16 weeks post-transplant. [Figure 20] Bone marrow reconstitution 16 weeks post-transplant using the experimental protocol shown in FIG. [Figure 21] Reconstitution of myeloid, B, and T lymphoid cells in peripheral blood and bone marrow at multiple time points using the experimental protocol shown in Figure 19. N=5 / group, data represent smallest to largest of 4 independent experiments. [Figure 22]Schematic diagram of transplantation studies to assess stem cell function of HSCs edited with sgRNAs from the gamma globin promoter region (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) compared with gRNAs from the erythroid-specific enhancer region of the BCL11A gene (sg-G1128; also referred to as sg1128). Cells were placed in culture for 24 hours after electroporation. Culture conditions before and after electroporation were StemSpan SFEM + IL6, SCF, TPO, Flt3L; 750 nM Compound 4. [Figure 23-1] Figure 23: Figure 23. Human engraftment and lineage analysis over 20 weeks in NSG mice. Figure 23A) Peripheral blood chimerism over 18 weeks; Figure 23B) Lineage distribution in peripheral blood at 18 weeks. Bone marrow analysis: Figure 23C) Bone marrow analysis of human cells at 9 weeks; Figure 23D) Human CD45+ engraftment and lineage distribution of human cells in bone marrow at 9 weeks; Figure 23E) Human CD45+ engraftment in bone marrow at 20 weeks post-engraftment; Figure 23F) Lineage distribution of engrafted cells in bone marrow at 20 weeks. [Figure 23-2] (As mentioned above.) [Figure 24-1] Figure 24: Figure 24A shows the engraftment efficiency of HSCs edited with sgRNAs homologous to the gamma-globin promoter region (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) compared to an sgRNA from the erythroid-specific enhancer region of the BCL11A gene (sg1128). Human cell engraftment in NSG mice at 8 weeks post-transplant is shown. N=10 / group, 3 independent experiments. Graph shows pooled data. Figure 24B shows the engraftment efficiency of HSCs edited with sgRNAs homologous to the gamma globin promoter region (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) compared with an sgRNA from the erythroid-specific enhancer region of the BCL11A gene (sg1128). Human cell engraftment in NSG mice at 20 weeks post-transplant is shown. N=10 / group, 3 independent experiments. Graph shows pooled data. [Figure 24-2](As mentioned above.) [Figure 25] Multilineage reconstitution of NSG mice transplanted with gene-edited CD34+ cells. N=10 / group, data from one representative experiment. [Figure 26] High editing efficiency was maintained before and after transplantation. "Pre-Xpt": Editing efficiency and indel patterns of individual sgRNAs in human CD34+ cells as measured by NGS before transplantation but at the time of editing. "8 weeks after transplantation (8 wks Post-Xpt)": Editing efficiency and indel patterns in human CD34+ cells 8 weeks after bone marrow transplantation in mice as measured by NGS. "20 weeks after transplantation (20 wks Post-Xpt)": Editing efficiency and indel patterns in human CD34+ cells 20 weeks after bone marrow transplantation in mice as measured by NGS. Electroporations were performed in triplicate per group; data from one representative experiment. As used in connection with this figure, "indel" refers to the sum of all indels less than 200 nt; "large deletion" refers to the deletion of sequence between the predicted HBG1 and HBG2 binding sites for each gRNA. [Figure 27] NGS analysis of CD34+ cells after editing with RNP. The sgRNA target-specific region is indicated on the x-axis. Figure 27A: NGS analysis of CD34+ cells 2 days after electroporation of RNP. Figure 27B: NGS analysis of whole bone marrow from an NSG mouse transplanted with edited bone marrow CD34+ cells 9 weeks after transplant (27B). Figure 27C: NGS analysis of whole bone marrow from an NSG mouse transplanted with edited bone marrow CD34+ cells 20 weeks after transplant. Insertional indels are shown in black, and deletional indels (excluding large deletions involving excision of the region between the combined HBG1 and HBG2 target sequences for gRNAs sg-G51, sg-G48, and sg-G67, respectively) are shown in gray. % total editing is represented by bar height. N=10, data shown as mean ± SEM from one independent experiment. [Figure 28]Gene-edited, long-term engrafted human HSCs were capable of producing increased levels of HbF during erythroid differentiation. Fifty thousand human CD34+ cells were sorted from the bone marrow of NSG mice engrafted for 8 or 20 weeks. Sorted cells were seeded in erythroid differentiation medium for 14–21 days. Mature erythrocytes in the culture were assayed for HbF expression, and the number of HbF+ cells was counted by flow cytometry. Mock controls represented CD34+ cells edited with Cas9 without gRNA and engrafted into NSG mice in the same manner as the gene-edited control group. N=10 / group, 3 independent experiments. [Figure 29] Off-target activity for HBG1 and / or HBG2 guide RNAs was assessed using a dsDNA oligo insertion method in Cas9-overexpressing HEK-293 cells. Detected on-target sites (open circles) and potential off-target sites (filled circles) are shown; the y-axis indicates the frequency of detection. All gRNAs were tested in the dgRNA format with targeting domains indicated by the CRxxxxxx identifier. [Figure 30] Off-target activity for HBG1 and / or HBG2 guide RNAs was assessed using a dsDNA oligo insertion method in Cas9-overexpressing HEK-293 cells. Detected on-target sites (open circles) and potential off-target sites (filled circles) are shown; the y-axis indicates the frequency of detection. gRNAs were tested in either dgRNA format with targeting domains indicated by CRxxxxx identifiers or sgRNA format with targeting domains indicated by Gxxxxxx identifiers. [Figure 31-1]Figure 31: Figure 31A shows CD34+ cell counts among cells derived from mobilized peripheral blood of a healthy individual at the time of gene editing. Cells were thawed on day 0 and cultured for 3 days before electroporation on day 3. CD34+ cell counts were determined by ISHAGE over 10 days after electroporation. Two independent experiments were performed in duplicate. Total N=5. The graph shows data from one experiment, expressed as mean ± SEM. Figure 31B shows CD34+ cell proliferation among cells derived from mobilized peripheral blood of a healthy individual at the time of gene editing. Cells were thawed on day 0 and cultured for 3 days before electroporation on day 3. Total mononuclear cell proliferation on days 3, 7, and 10 after editing. Two independent experiments were performed in duplicate. Total N=5. The graph shows data from one experiment, expressed as mean ± SEM. Figure 31C shows CD34+ cell viability among cells derived from mobilized peripheral blood of a healthy individual at the time of gene editing. Cells were thawed on day 0 and cultured for 3 days before electroporation on day 3. Mononuclear cell viability at the same time points after editing. Two independent experiments were performed in duplicate. Total N=5. Graphs show data from one experiment as mean ± SEM. [Figure 31-2] (As mentioned above.) [Figure 31-3] (As mentioned above.) [Figure 32-1]Figure 32: Figure 32A shows the editing efficiency of sg1128 and sg0067 in CD34+ cells mobilized from the peripheral blood of a healthy individual. The percentage of indels captured by NGS during editing with sg1128 (targeting the BCL11A+58 region of ESH) and sg0067 (targeting the HbG-1 and HbG-2 gene cluster) is shown. Because only small indels were generated by this sgRNA, the graph also shows the overall editing efficiency for sg1128. Six or more independent experiments were performed in duplicate or triplicate (n = 2–3 per experiment). Figure 32B shows the editing efficiency of sg1128 and sg0067 in CD34+ cells mobilized from the peripheral blood of a healthy individual. A. Percentage of indels captured by NGS during editing with sg0067 (targeting the HbG-1 and HbG-2 gene clusters). The overall editing efficiency and editing pattern of sg0067 are shown. The editing pattern of sg0067 consists of a large 5 kb deletion (indicated by the black bar) and smaller indels (indicated by the gray bar). Six or more independent experiments were performed in duplicate or triplicate (n = 2–3 per experiment). [Figure 32-2] (As mentioned above.) [Figure 33] Fold change in gamma-globin transcripts in erythroid cells from patient samples upon CRISPR knockdown of BCL11A or indel / deletion formation in the HBG1 / 2 region. CD34+ cells derived from mobilized peripheral blood of healthy donors were CRISPR-edited and differentiated in vitro toward the erythroid lineage as described in the previous procedure. On day 11 of erythroid differentiation, cells were harvested from culture and subjected to qPCR to measure gamma-globin and b-globin transcripts, normalized to GAPDH. Each experiment was performed twice independently in duplicate (n = 2–3 per experiment). Data represent the mean ± SEM of pooled donors from one study. [Figure 34]Enumeration of HbF+ cells from a healthy individual at the time of gene editing. CD34+ cells derived from mobilized peripheral blood of a healthy donor were edited using CRISPR and differentiated in vitro toward the erythroid lineage as described in the previous procedure. On day 10 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody and HbF+ cells were counted by flow cytometry. Each experiment was performed independently and in duplicate (n = 2-3 per experiment). Data represent the mean ± SD from one experiment. [Figure 35][Figure 35A] Proliferation and viability of CD34+ cells derived from peripheral blood of an individual with sickle cell disease upon gene editing. Cells were cultured for 6-10 days before electroporation. D0 refers to the day of electroporation. Absolute numbers of CD34+ cells by ISHAGE over 10 days after electroporation are shown. N=4, data represent mean ± SEM. Four independent experiments were performed in duplicate. Bars represent mock, sg1128, and sg0067 at each time point, from left to right. [Figure 35B] Proliferation and viability of CD34+ cells derived from peripheral blood of an individual with sickle cell disease upon gene editing. Cells were cultured for 6-10 days before electroporation. D0 refers to the day of electroporation. Percentages of CD34+ cells by ISHAGE over 10 days after electroporation are shown. N=4, data represent mean ± SEM. Four independent experiments were performed in duplicate. Bars, from left to right, represent mock, sg1128, and sg0067 at each time point. [Figure 35C] Proliferation and viability of CD34+ cells derived from peripheral blood of individuals with sickle cell disease upon gene editing. Cells were cultured for 6 to 10 days before electroporation. D0 refers to the day of electroporation. Total mononuclear cell proliferation at days 3, 7, and 10 after editing is shown. N=4, data represent mean ± SEM. Four independent experiments were performed in duplicate. Bars, from left to right, represent mock, sg1128, and sg0067 at each time point. [Figure 35D] Proliferation and viability of CD34+ cells derived from peripheral blood of individuals with sickle cell disease upon gene editing. Cells were cultured for 6 to 10 days before electroporation. D0 refers to the day of electroporation. Mononuclear cell viability at days 3, 7, and 10 after editing is shown. N=4, data represent mean ± SEM. Four independent experiments were performed in duplicate. Bars are mock, sg1128, and sg0067 from left to right at each time point. [Figure 36]Editing efficiency of sg1128 and sg0067 in CD34+ cells from sickle cell disease patient samples. The editing pattern of sg0067 is shown by the sum of large deletions (gray) and small indels (black). Data represent the mean ± SEM of four independent editing experiments performed in duplicate using CD34+ cells from four different sickle cell disease patients (SCD1-4). [Figure 37] In vitro multilineage differentiation potential of HSPCs as measured by colony-forming unit assay. BFU-E = burst-forming unit, erythroid; CFU-GM = colony-forming unit, granulocyte, monocyte; CFU-GEMM = colony-forming unit, granulocyte, erythroid, monocyte, megakaryocytic. Graph shows three independent experiments from CD34+ cells from three different sickle cell disease patients (SCD1-3). Experiments were performed in triplicate. Data represent mean ± SEM. [Figure 38] Fold change in g-globin transcripts in erythroid cells from patient samples upon CRISPR knockdown of BCL11A or indel / deletion formation in the g-globin gene cluster. CD34+ cells from three sickle cell disease patients (SCD1-3) were edited by CRISPR and differentiated in vitro toward the erythroid lineage as described in the Examples. On day 11 of erythroid differentiation, cells were harvested from culture and subjected to qPCR to measure g-globin and b-globin transcripts, normalizing to GAPDH. Experiments were performed in duplicate. Data represent the mean ± SEM of pooled donors. [Figure 39]Enumeration of HbF+ cells in sickle cell disease patient samples during gene editing. CD34+ cells from three sickle cell disease patients (SCD1-3) were edited using CRISPR and differentiated in vitro toward the erythroid lineage as described in the Examples. On days 11, 14, and 21 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody and HbF+ cells were counted by flow cytometry. Experiments were performed in duplicate. Data represent mean ± SEM. Results from SCD1-3 are shown at day 11; results from SCD-1 and SCD-2 are shown at day 14; and results from SCD-2 and SCD-3 are shown at day 21. [Figure 40] Measurement of fetal hemoglobin expression in gene-edited sickle cell disease patient samples by flow cytometry. CD34+ cells from three sickle cell disease patients (SCD1-3) were edited using CRISPR and differentiated in vitro toward the erythroid lineage as described in the Examples. On days 11, 14, and 21 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody and the HbF expression intensity of each cell was measured by flow cytometry. Experiments were performed in duplicate. Data represent the mean ± SEM. MFI = mean fluorescence intensity. Results from SCD1-3 are shown at day 11; results from SCD-1 and SCD-2 are shown at day 14; and results from SCD-2 and SCD-3 are shown at day 21. [Figure 41]Counting sickle cell counts relative to normal cells after CRISPR editing of patient samples. CD34+ cells from three sickle cell disease patients (SCD1-3) were edited with CRISPR and differentiated in vitro toward the erythroid lineage as described in the previous procedure. On day 21 of erythroid differentiation, cells were placed in a 50% hypoxia chamber for 4 days, fixed, and then subjected to single-cell imaging flow cytometry. Figure 41A (left panel) shows the change in the number of sickle cell counts in the gene-edited patient sample, as counted by single-cell imaging. Figure 41B (right panel) shows the change in the number of normal cells in the patient sample after gene editing, as counted by single-cell imaging. Three independent experiments were performed, each with duplicates. 40,000 single-cell images from each patient were counted. Graphs show mean ± SEM from pooled data. DETAILED DESCRIPTION OF THE INVENTION

[0120] definition The terms "CRISPR system," "Cas system," or "CRISPR / Cas system" The "system" acts together to target the RNA-guided nuclease or other effector molecule at the target sequence. RNA-guided nucleases or nucleases necessary and sufficient to induce and achieve nucleic acid modification by the molecule. refers to a set of molecules that includes a gRNA molecule and other effector molecules. The CRISPR system combines gRNA and a Cas protein, such as the Cas9 protein. Such systems comprising Cas9 or modified Cas9 molecules are referred to herein as "Cas9 In one example, the gRN The A molecule and the Cas molecule can complex together to form a ribonucleoprotein (RNP) complex.

[0121] The terms "guide RNA," "guide RNA molecule," "gRNA molecule," or "gRNA" refer to Used interchangeably, RNA-guided nucleases or other effector molecules (typically Nucleic acid molecules that promote the specific targeting of gRNA molecules (complexed with gRNA molecules) to target sequences. In some embodiments, the induction refers to a set of genes that are linked to DNA (e.g., a set of genes). through hybridization (e.g., via the gRNA targeting domain) and A portion of the A molecule is attached to an RNA-guided nuclease or other effector molecule (e.g., This is achieved by binding (at least via gRNA tracr). In this context, a gRNA molecule consists of a single contiguous polynucleotide molecule, and is referred to herein as These may also be referred to as "single guide RNAs" or "sgRNAs." In other embodiments, gRNA molecules have the ability to associate with themselves, usually through hybridization It consists of multiple, usually two, polynucleotide molecules, and is referred to herein as a "dual guide" gRNA molecules are referred to as "gRNA" or "dgRNA." More details about gRNA molecules are provided below. In the embodiments, the targeting domain and the tracr are generally included. In other embodiments, the targeting domain and tracr are located on a single polynucleotide. In the present invention, the targeting domain and tracr are located on separate polynucleotides.

[0122] The term "targeting domain," when used in reference to a gRNA, refers to a target sequence. sequence that recognizes, e.g., is complementary to, a target sequence within the nucleic acid of a cell, e.g., a gene is a portion of a gRNA molecule that is

[0123] The term "crRNA" when used in reference to a gRNA molecule refers to a targeting domain. gRN, which contains the main and the region that interacts with tracr to form the flagpole region It is part of the A molecule.

[0124] The term "target sequence" refers to a nucleic acid sequence that is complementary, e.g., perfectly complementary, to a gRNA targeting domain. In an embodiment, the target sequence is located on genomic DNA. In some embodiments, the target sequence is targeted to a protein with nuclease or other effector activity. Protospacer adjacent motif (PAM) sequences recognized by, for example, Cas9 adjacent to the PAM sequence (either on the same strand or on the complementary strand of DNA) that is recognized by In embodiments, the target sequence is a gene or gene sequence that affects the expression of a globin gene. Within a gene locus, for example, a gene affecting the expression of beta-globin or fetal hemoglobin (HbF) In an embodiment, the target sequence is a non-deleted HP. Target sequences within the FH region, e.g., the HBG1 and / or HBG2 promoter regions It's inside.

[0125] The term "flagpole," as used herein in reference to a gRNA molecule, refers to a The RNA and tracr refers to the portion of the gRNA that binds or hybridizes to each other. vinegar.

[0126] The term "tracr" as used herein in reference to a gRNA molecule refers to a nuclease In some embodiments, the term "gRNA" refers to the portion of the gRNA that binds to a targeting enzyme or other effector molecule. tracr comprises a nucleic acid sequence that specifically binds to Cas9. The cr contains a nucleic acid sequence that forms part of the flagpole.

[0127] The term "Cas9" or "Cas9 molecule" refers to a bacterial type II CRIS involved in DNA cleavage. It refers to the enzyme of the PR / Cas system. Cas9 contains wild-type proteins and their functional and non-functional mutants are also included. In embodiments, Cas9 is used to transform Streptococcus pyogenes (S The Cas9 gene is from B. pyogenes.

[0128] The term "complementary" when used in reference to nucleic acids refers to the bases A and T or U, and G and C. The term complementary refers to perfect complementarity, i.e., complete complementarity across the entire reference sequence. nucleic acid molecules in which A pairs with T or U and G pairs with C, and at least 8 Refers to 0%, 85%, 90%, 95%, and 99% complementary molecules.

[0129] "Template nucleic acid" when used in connection with homologous repair or homologous recombination refers to a nucleic acid fragment that is Site modified by CRISPR system donor sequence for gene repair (insertion) at position It refers to the nucleic acid inserted into a

[0130] "Indels," as the term is used herein, refer to a gene that is a gene encoding a gene encoding a gRNA molecule. A compound, e.g., a CRISPR system, produces one or more nucleic acids compared to a reference nucleic acid. Nucleotide insertion, one or more nucleotide deletions, or a combination of nucleotide insertions and deletions Indels refer to nucleic acids that contain a sequence similar to that described above. Indels are nucleic acids that have undergone exposure to a composition containing a gRNA molecule, e.g., For example, the location of the indel can be determined by sequencing using NGS. An indel is one that is located about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide away from the reference site. containing at least one insertion or deletion within the range of the reference site, or a portion of the reference site or overlapping all (e.g., gRNA molecules, e.g., gRNA molecules described herein) overlapping or separated from the region complementary to the targeting domain of the RNA molecule at least one within 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides If the targeting domain of the gRNA molecule is a reference site (e.g., containing a single insertion or deletion), In some embodiments, an indel is "at or near" a site (or complementary site). is, for example, greater than about 1 kb, greater than about 2 kb, greater than about 3 kb, greater than about 4 kb, greater than about 5 kb, or greater than about 6 kb. In embodiments, the large deletion is a large deletion comprising more than about 10 kb of nucleic acid. The 5' end, the 3' end, or both the 5' and 3' ends of the gRNA molecule described herein In an embodiment, the large deletion is located at or near the target sequence of HBG. 1. A target sequence for a gRNA molecule, e.g., as described herein, located within the promoter region and a gRNA molecule, e.g., as described herein, positioned within the HBG2 promoter region. It contains approximately 4.9 kb of DNA located between the target sequence.

[0131] "Indel pattern," as the term is used herein, refers to a pattern of indels in a gRNA molecule. In certain embodiments, the indel refers to a set of indels that arise after exposure to a composition comprising The pattern consists of the top three indels based on frequency of occurrence. The indel pattern consists of the top five indels based on frequency of occurrence. In this state, indel patterns occur at a frequency higher than approximately 1% of all sequencing reads. In one embodiment, the indel pattern consists of indels present in the entire sequence. In some embodiments, the indels are present at a frequency greater than about 5% relative to the single read. In this study, the indel pattern was calculated based on the total number of indel sequencing reads (i.e., unmodified reference sequences). Indels present at a frequency higher than about 10% relative to the reads (reads not consisting of the reference nucleic acid sequence) In one embodiment, the indel pattern consists of the top 5 most frequently observed The indel pattern may include any three of the indels described herein, for example. The method includes, for example, sequencing cells of a population of cells exposed to a gRNA molecule. It can be determined by

[0132] "Off-target indels," as the term is used herein, refer to gRN A refers to an indel located at or near a site other than the target sequence in the targeting domain of a molecule. Such sites may be, for example, 1, 2, 3, 4, 5 or more relative to the sequence of the targeting domain of the gRNA. In an exemplary embodiment, such a site may contain more than one mismatched nucleotide. using targeted sequencing of predicted off-target sites in silico, or The gRNAs described herein are detected by insertion methods known in the art. Examples of off-target indels include those in the HBG1 and / or HBG2 promoter regions. In an exemplary embodiment, an indel is formed in a sequence outside of the off-target region. The target indel is formed within the sequence of the gene, for example within the coding sequence of the gene.

[0133] The terms "a" and "an" refer to one of the grammatical referents of the article. or more than one (i.e., at least one). By way of example, "an element" refers to one element or It means two or more elements.

[0134] The term "about" when referring to a measurable value, such as an amount, duration, or the like, means a certain number of degrees from the specified value. ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1% or in some cases ±0.1% variation (such variation is suitable for practicing the methods of the present disclosure). It is intended to encompass (as)

[0135] The term "antigen" or "Ag" refers to a molecule that provokes an immune response. This may involve either antibody production or activation of specific immunocompetent cells, or both. Any macromolecule can be an antigen, including virtually any protein or peptide. It will be understood that antigens may be derived from recombinant or genomic DNA. Those skilled in the art will therefore be able to identify nucleotide sequences encoding proteins that will generate an immune response. or any DNA containing a partial nucleotide sequence is as that term is used herein. It will be understood that the present invention encodes an "antigen" as it is expressed in a nucleic acid sequence. It is understood that a gene need not be encoded solely by its full-length nucleotide sequence. The present invention includes, but is not limited to, partial nucleotide sequences of two or more genes. The present invention also includes the use of nucleotide sequences that produce the desired immune response. It is readily apparent that the sequences may be arranged in various combinations to encode polypeptides of interest. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be understood that antigens can be synthetic or obtained from biological samples, or can be prepared by polyclonal antibodies. It is readily apparent that the biological sample may be a macromolecule other than a peptide. may include, but are not limited to, tissue samples, bodily fluids containing cells or other biological components .

[0136] The term "autologous" refers to any gene derived from the same individual into which it is later reintroduced. Refers to the material.

[0137] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into whom the material is introduced. Two or more individuals are different from each other when the genes at one or more loci are not identical. In some embodiments, allogeneic origin from individuals of the same species. The source material may be sufficiently genetically distinct to interact as an antigen.

[0138] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0139] "Derived from," as that term is used herein, refers to a molecule that is derived from a first molecule and a second molecule. This generally refers to the structural relationship between a first molecule and a second molecule. Similarity refers to the process or source limitation of a first molecule derived from a second molecule. Or does not include.

[0140] The term "encode" refers to the sequence of a sequence in a polynucleotide, such as a gene, cDNA, or mRNA. The specific nucleotide sequence is a defined nucleotide sequence (e.g., rRNA, tR NA and mRNA) or a defined amino acid sequence and the organisms that result from it as templates for the synthesis of other polymers and macromolecules in biological processes with biological properties Thus, a gene, cDNA, or RNA refers to a specific characteristic that plays a role in the Proteins are produced in cells or other biological systems by transcription and translation of mRNA corresponding to a gene. When a protein is produced, its nucleotide sequence encodes the protein. and the coding strand, which is usually provided in the sequence listing, as a template for transcription of the gene or cDNA. Both the coding and non-coding strands used encode the protein or other product of that gene or cDNA. This may be referred to as coding.

[0141] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are degenerate versions of each other. A protein is a sequence of nucleotides that all encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" includes the sequence of the nucleotides that encode that protein. Insofar as the nucleotide sequence may, in some versions, contain one or more introns, In the present invention, introns may also be included.

[0142] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and are used interchangeably herein. The compounds, formulations, materials, or compositions as described herein are intended to achieve a particular biological result. Refers to an effective amount.

[0143] The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system. Refers to the fee.

[0144] The term "exogenous" refers to something introduced from or produced outside an organism, cell, tissue, or system. refers to any material produced from

[0145] The term "expression" refers to the transcription of a particular nucleotide sequence driven by a promoter. and / or refers to translation.

[0146] The term "transfer vector" refers to a composition containing an isolated nucleic acid that is used to transfer the nucleic acid to the interior of a cell. Refers to compositions that can be used to deliver isolated nucleic acids, including but not limited to linear polynucleotides, Polynucleotides, plasmids, and viruses associated with ionic or amphiphilic compounds Many vectors are known in the art, including those described herein. A "transgenic vector" includes an autonomously replicating plasmid or virus. This term is used to refer to, for example, Non-plastic polymers that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes, The term "viral" should be construed to further include smids and non-viral compounds. Examples of transfer vectors include, but are not limited to, adenovirus vectors, adenovirus vectors, Examples include virus-associated vectors, retrovirus vectors, and lentivirus vectors. can be done.

[0147] The term "expression vector" refers to an expression control vector operably linked to a nucleotide sequence to be expressed. An expression vector refers to a vector containing a recombinant polynucleotide containing a sequence. It contains sufficient cis-acting elements for expression. Other elements for expression are supplied by the host cell. The expression vector can be a recombinant polynucleotide. Cosmids, plasmids (e.g., naked or contained in liposomes) incorporating the peptide viruses (e.g., lentiviruses, retroviruses, adenoviruses, and These include all those known in the art, including vectors such as avian encephalopathy virus (AVV) and adeno-associated viruses.

[0148] The terms "homology" or "identity" refer to the relationship between two polymer molecules, e.g., two DNA molecules or is a sequence between two nucleic acid molecules, such as two RNA molecules, or between two polypeptide molecules. The sequence identity of the subunits in both molecules is the same. For example, when each position on two DNA molecules is occupied by an adduct subunit, If the position is occupied by a nucleotide, then they are homologous or identical at that position. Homology is a direct function of the number of matching or homologous positions. For example, Half of the positions in the polymer are homologous (e.g., 5 positions in a 10-subunit long polymer). If 90% of the positions (e.g., 10 9) are matched or homologous, then the two sequences are 90% homologous.

[0149] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is "isolated." It is not "in the state of being" but is partly or completely separated from the coexisting materials in its natural state. The nucleic acid or peptide is "isolated." An isolated nucleic acid or protein is one that has been substantially purified. It can exist in a modified form or in a non-native environment, such as a host cell. can.

[0150] The terms "operably linked" or "transcriptional control" refer to the link between a regulatory sequence and a heterologous nucleic acid sequence. For example, a first nucleic acid sequence may be linked to a second nucleic acid sequence to provide for expression of the latter in the context of the first nucleic acid sequence. When placed in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence functions in a functional relationship with the second nucleic acid sequence. For example, a promoter may be used to influence the transcription or expression of a coding sequence. A promoter is operably linked to a coding sequence when it controls the expression of the gene. The DNA sequences may be adjacent to each other, for example, as needed to join two protein coding regions. If necessary, they are in the same reading frame.

[0151] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous ( iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0152] The term "nucleic acid" or "polynucleotide" refers to a nucleic acid in either single-stranded or double-stranded form. It refers to DNA or RNA and its polymers. Unless otherwise specified, the term includes nucleic acids that have similar binding properties to the reference nucleic acid and that are naturally occurring. Nucleic acids containing known analogues of natural nucleotides that are metabolized in a manner similar to nucleotides are also included. Unless otherwise indicated, a particular nucleic acid sequence also implicitly refers to its conservatively modified forms. modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences Specifically, degenerate codon substitutions include one or more Mixed base and / or deoxyinosine residues in the third position of selected (or all) codons This can be achieved by creating a sequence in which the nucleotide sequence is substituted with nucleotides (Batzer et al. ,Nucleic Acid Res.19:5081(1991);Ohtsuka et al., J.Biol.Chem.260:2605-2608(1985); and Rossolini et al., Mol. Cell. Probes 8:91-9 8(1994)).

[0153] The terms "peptide," "polypeptide," and "protein" are used interchangeably, and A compound consisting of amino acid residues covalently linked by tidyl bonds. A protein or peptide must contain at least two amino acids and the structure of the protein There is no limit imposed on the maximum number of amino acids that a polypeptide or peptide sequence can contain. A peptide is any molecule containing two or more amino acids joined together by peptide bonds. As used herein, the term includes peptides or proteins of the In the field, short chains, also commonly referred to as peptides, oligopeptides and oligomers, are used. This term refers to both the many types of longer chains generally referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, and the like. Polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides Polypeptides include, among others, naturally occurring peptides, derivatives, analogs, and fusion proteins. The present invention includes a recombinant peptide, a recombinant peptide, or a combination thereof.

[0154] The term "promoter" refers to a promoter or promoter sequence required to initiate the specific transcription of a polynucleotide sequence. A DNA sequence that is recognized by the synthetic machinery of a cell or by introduced synthetic machinery.

[0155] The term "promoter / regulatory sequence" refers to a sequence operably linked to that promoter / regulatory sequence. This refers to a nucleic acid sequence required for expression of a gene product derived from a core promoter. In other cases, this sequence may be an enhancer sequence and a sequence necessary for expression of the gene product. The promoter / regulatory sequence may, for example, direct the expression of a gene product. It may be expressed in a tissue-specific manner.

[0156] The term "constitutive" promoter refers to a polynucleotide that encodes or specifies a gene product. When operably linked to a nucleotide, it is capable of surviving in cells under most or all physiological conditions of the cell. It refers to a nucleotide sequence that causes the production of a gene product in the cell.

[0157] The term "inducible" promoter refers to a polynucleotide that encodes or specifies a gene product. an inducer that, when operably linked to a nucleotide, substantially corresponds to a promoter A nucleotide sequence that causes the production of a gene product in a cell only if the nucleotide sequence is present in the cell. Point to the column.

[0158] The term "tissue-specific" promoter refers to a promoter that encodes or is specified by a gene. In effect, the cell responds to the promoter when the promoter is operably linked to a polynucleotide that responds to the promoter. Nucleotides that cause the production of a gene product in cells only if they are cells of the corresponding tissue type. Refers to the octide sequence.

[0159] As used herein in reference to messenger RNA (mRNA), 5' capped RNA cap, RNA 7-methylguanosine cap or RNA m7G cap (also called the "front" or 5' end of eukaryotic messenger RNAs immediately after transcription initiation) The 5' cap is a modified guanine nucleotide added to the first transcribed nucleotide. Its presence determines the degree of recognition by the ribosome and the ability to react with RNases. Capping is important for protecting the nuclei from transcription. Capping is coupled to transcription, and each influences the other. Immediately after the start of transcription, a co-transcriptional sequence is formed at the 5' end of the synthesized mRNA. The cap-synthesizing complex associated with RNA polymerase binds to the m It catalyzes the chemical reactions required for RNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety is used to regulate the functions of mRNA, such as its stability or translation efficiency. It can be modified.

[0160] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro. A, preferably mRNA. Generally, in vitro transcribed RNA is expressed as an in vitro transcription vector. In vitro transcription vectors are used to generate in vitro transcribed RNA. It includes a template.

[0161] As used herein, "poly(A)" refers to the polyadenylation that is attached to mRNA. A preferred embodiment of the construct for transient expression is In this case, poly A is 50 to 5000 (SEQ ID NO: 190), preferably more than 64, more preferably Preferably, the poly(A) sequence is greater than 100, and most preferably greater than 300 or 400. chemically or enzymatically to modulate mRNA functions such as localization, stability, or translation efficiency It can be modified.

[0162] As used herein, "polyadenylation" refers to the addition of polyadenylation to a messenger RNA molecule. It refers to the covalent attachment of a liadenylyl moiety or a modified variant thereof. In eukaryotes, most The messenger RNA (mRNA) molecule is polyadenylated at the 3' end. The tail is added to the pre-mRNA by the action of the enzyme polyadenylate polymerase Adenine nucleotides are long sequences (often hundreds) of adenine nucleotides that form a chain. In higher eukaryotes, A poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins bound to it act to digest the mRNA by exonuclease. Polyadenylation is the process that terminates transcription, protecting mRNA from degradation by ribosomal enzymes. Polyadenylation is also important for the transport and translation of DNA into RNA. After transcription is complete, RNA polymerase The mRNA strand is cleaved by the action of an endonuclease complex associated with the cleavage site. mRNA is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After A is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0163] As used herein, "transient" refers to a condition that lasts for a period of hours, days, or weeks. Refers to the expression of a non-integrated transgene, the period during which it is expressed without being integrated into the genome of the host cell. Expression period of genes when incorporated into a plasmid or contained in a stable plasmid replicon is shorter than.

[0164] As used herein, the terms "treat," "treatment," and "treating" refer to one or more therapies (e.g., gRNA molecules, CRISPR systems, or modified cells of the invention) Disorders resulting from the administration of one or more therapeutic agents, such as hemoglobinopathies Reduction or amelioration of the progression, severity and / or duration of, or disorders, such as hemoglobinopathies The term "improvement of one or more symptoms (preferably one or more discernible symptoms) of a specific condition." In embodiments, the terms "treat," "treatment," and "treating" are used in conjunction with patient-perceived improvement in at least one measurable physical parameter of a hemoglobinopathy disorder In another embodiment, the terms "treat", "treatment" and "treating" refer to Inhibition of the progression of the disorder physically, e.g., by stabilization of discernible symptoms; physiologically, e.g., In other embodiments, the present invention refers to either or both of the inhibition of the inflammatory bowel disease and the stabilization of physiological parameters. In this context, the terms "treat," "treatment," and "treating" refer to the treatment of hemoglobinopathies, e.g. Refers to the reduction or stabilization of symptoms of sickle cell disease or beta-thalassemia.

[0165] The term "signal transduction pathway" refers to the transmission of a signal from one part of a cell to another part of a cell. The term "cell surface" refers to the biochemical relationships between various signaling molecules that play a role in "Receptors" are molecules and molecular complexes that receive signals and transmit them across the membrane of a cell. Including the body.

[0166] The term "subject" refers to a living organism in which an immune response can be generated (e.g., a mammal). , humans).

[0167] The term "substantially purified" cells refers to cells that are essentially free of other cell types. A qualitatively purified cell is one that is distinct from other cells with which it is normally associated in its naturally occurring state. The term "substantially purified" also refers to cells that have been separated into different cell types. In some instances, a population of substantially purified cells may be In other instances, the term simply refers to a homogeneous population of cells in their natural state as they naturally occur. In some embodiments, these cells are In other embodiments, the cells are not cultured in vitro.

[0168] The term "therapeutic" as used herein means treatment. A therapeutic effect is a disease This is achieved by reducing, suppressing, ameliorating, or eradicating the condition.

[0169] The term "prevention" as used herein refers to the prevention or protective treatment of a disease or disease state. It means treatment.

[0170] The terms "transfected" or "transformed" or "transduced" mean Refers to the process of transferring or introducing exogenous nucleic acids and / or proteins into a host cell A cell that has been "transfected" or "transformed" or "transduced" is Transfected, transformed, or transfected with exogenous nucleic acids and / or proteins The cell includes the primary subject cell and its progeny.

[0171] The term "specifically binds" refers to the ability of a binding partner (e.g., a protein) present in a sample to bind to the target protein. or nucleic acid) but do not substantially bind to other molecules in the sample. It refers to a molecule that does not recognize or bind to.

[0172] The term "bioequivalent" means a compound that is equivalent to the effect produced by a reference dose or a reference amount of a reference compound. It refers to the amount of a drug other than the reference compound that is required to produce an equivalent effect.

[0173] "Refractory," as used herein, refers to a disease that does not respond to treatment, e.g., a disorder characterized by abnormal hemoglobin levels. In an embodiment, refractory hemoglobinopathy is a condition that occurs before or after treatment is initiated. In another embodiment, the refractory hemoglobinopathy is refractory to treatment at that time. Refractory hemoglobinopathy is also called resistant hemoglobinopathy. will be done.

[0174] "Recurrent" as used herein refers to a condition that develops after a period of improvement, e.g., after treatment with a different therapy, e.g., Hemoglobinopathy treatment is recommended for patients with hemoglobinopathy or other disorders (e.g., hemoglobinopathy) after prior hemoglobinopathy treatment. globinopathy) or the return of signs and symptoms of the disease.

[0175] Ranges: Throughout this disclosure, various aspects of this invention may be presented in a range format. It will be understood that the formal description is merely for convenience and brevity. and should not be construed as inflexibly limiting the scope of the present invention. Therefore, the description of a range includes all the possible subranges as well as individual numerical values ​​within that range. For example, a range such as 1 to 6 must be deemed to be a specific disclosure. , 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and other partial ranges within that range Specifically disclosed are individual numbers in the range of 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Another example is a range such as 95-99% identity. The range includes those with 95%, 96%, 97%, 98% or 99% identity. , and 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 9 Subranges such as 8-99% identity are included. This applies regardless of the breadth of the range. can be.

[0176] The term "BCL11a" refers to the RNA polymerase II core promoter-proximal region sequence-specific B-cell lymphoma / leukemia 11A, a specific DNA-binding protein, and This refers to the gene that encodes the C2H2 type, along with all introns and exons. It encodes a zinc finger protein. BCL11A suppresses fetal hemoglobin production. BCL11a is known to play a role in the regulation of B-cell CLL / lymphoma. 11A (zinc finger protein), CTIP1, EVI9, ecotropic virus integration site 9 protein homolog, COUP-TF interacting protein 1, zinc phosphatase inger protein 856, KIAA1809, BCL-11A, ZNF856, EVI- Also known as B-cell CLL / lymphoma 11A, BLC11a, and B-cell CLL / lymphoma 11B. All isoforms and splice variants are included. The human gene maps to chromosomal location 2p16.1 (Ensembl). Human and mouse amino acid and nucleic acid sequences are available from GenBank, UniProt, and Swiss Public databases such as .Prot and the human BCL11a genome can be referenced. The sequence can be found in GenBank as NC_000002.12. The CL11a gene refers to this genomic location, including all introns and exons. There are multiple known isotypes of CL11a.

[0177] The sequence of the mRNA encoding human BCL11a isoform 1 is NM_02289 3 can be referred to.

[0178] The peptide sequence of isoform 1 of human BCL11a is: [ka]

[0179] The sequences of other BCL11a protein isoforms are provided below. Isoform 2: Q9H165-2 Isoform 3: Q9H165-3 Isoform 4: Q9H165-4 Isoform 5: Q9H165-5 Isoform 6: Q9H165-6

[0180] As used herein, the human BCL11a protein includes the BCL11a protein over its entire length. BCL11a isoforms 1–6 and at least approximately 70%, 71%, 72%, 73%, and 7 4%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Proteins are also encompassed, where such proteins still exhibit at least some of the functions of BCL11a. Have one.

[0181] The term "globin locus" as used herein refers to embryonic (ε), fetal (γ) and A(γ)), ​​adult globin genes (γ and β), locus control region and DNase I This refers to the region of human chromosome 11 that contains the gene for the hypersensitive site.

[0182] The term "complementary" when used in reference to nucleic acids refers to the bases A and T or U, and G and C. The term complementary refers to perfect complementarity, i.e., complete complementarity across the entire reference sequence. nucleic acid molecules in which A pairs with T or U and G pairs with C, and at least 8 Refers to 0%, 85%, 90%, 95%, and 99% complementary molecules.

[0183] The term "non-deficient HPFH" refers to the condition resulting in hereditary hyperfetal hemoglobinemia and adult erythrocytic leukemia. insertion or deletion of one or more nucleotides, characterized by an increase in fetal hemoglobin in the cells In an exemplary embodiment, a non-deleted HPFH refers to a mutation that does not include a deletion. n and Oski's Hematology and Oncology of Infancy and Childhood, 8 th Ed., 2015, Orkin SH, Fisher DE, Look T, Lux SE, Ginsburg D, N athan DG, Eds., Elsevier Saunders (the entire contents of this Mutations described in Tables 21-5, which are incorporated herein by reference, e.g., mutations described in Tables 21-5 The term "non-deleted HPFH region" refers to a non-deleted HPFH mutation. In an exemplary embodiment, the term "deleted HPFH region" refers to a genomic site located at or near the genomic site. The region is the nucleic acid sequence of the HBG1 promoter region (Chr11:5,249,833 to Chr 11:5,250,237, hg38;-strand), the nucleic acid sequence of the HBG2 promoter region ( Chr11:5,254,738~Chr11:5,255,164, hg38;-chain) In an exemplary embodiment, the non-deleted HPFH region is N athan and Oski's Hematology and Oncology of Infancy and Childhood, 8 th Ed.,2015,O rkin SH, Fisher DE, Look T, Lux SE, Ginsburg D, Nathan DG, Eds., Elsevier Saunders The present invention includes one or more of the non-deleted HPFHs (e.g., as described in Table 21-5 therein). In one embodiment, the non-deleted HPFH region is chr11:5,250,094-5,2 50,237, -strand, nucleic acid sequence in hg38; or chr11:5,255,022 ~5,255,164, -strand, nucleic acid sequence in hg38; or chr11:5,249 ,833 to 5,249,927, -strand, nucleic acid sequence in hg38; or chr11:5 ,254,738 to 5,254,851, -strand, nucleic acid sequence in hg38; or chr 11:5,250,139 to 5,250,237, -strand, nucleic acid sequence in hg38; or is a combination of these.

[0184] "BCL11a enhancer," as the term is used herein, refers to a BCL11a enhancer. It refers to a nucleic acid sequence that affects, e.g., enhances, the expression or function of L11a. Bauer et al.,Science,vol.342,2013,pp.2 See, e.g., BCL11a enhancers are expressed in certain cell types, For example, it may be possible that BCL11a acts only in cells of the erythroid lineage. An example of a hanser is the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene (e.g., For example, the location recorded in hg38 is +55:Chr2:60497676~6 0498941;+58:Chr2:60494251~60495546;+62:C hr2:60490409-60491734 or the nucleic acid corresponding thereto) In one embodiment, the BCL11a enhancer is located in an exon of the BCL11a gene. In one embodiment, the nucleic acid sequence is the +62 region of the nucleic acid sequence between exon 2 and exon 3 of BC The L11a enhancer is located between exons 2 and 3 of the BCL11a gene. In one embodiment, the BCL11a enhancer is located in the +58 region of the BC It is the +55 region of the nucleic acid sequence located between exon 2 and exon 3 of the L11a gene.

[0185] The terms "hematopoietic stem and progenitor cells" or "HSPCs" are used interchangeably and refer to hematopoietic stem cells ( It refers to a population of cells that contains both hematopoietic progenitor cells ("HSCs") and hematopoietic progenitor cells ("HPCs"). In an exemplary embodiment, the HSPCs are characterized as CD34+. In another exemplary embodiment, HSPCs are isolated from peripheral blood. In another exemplary embodiment, the HSPCs are isolated from umbilical cord blood. In this study, HSPCs were characterized as CD34+ / CD38- / CD90+ / CD45RA- In an embodiment, the HSPCs are CD34+ / CD90+ / CD49f+ cells. In embodiments, HSPCs are characterized as CD34+ cells. In embodiments, HSPCs are characterized as CD34+ / CD90+ cells. In an embodiment, the HSPCs are CD34+ / CD90+ / CD45RA- cells. It can be characterized as:

[0186] "Stem cell expanding agent," as used herein, refers to a cell, e.g., HSPC, HSC, and and / or cause HPCs to proliferate, e.g., increase in number, at a faster rate than the same cell type without the agent. In an exemplary embodiment, the stem cell proliferation agent is an aryl hydrocarbon receptor. Further examples of stem cell proliferation agents are provided below. Thus, proliferation, eg, an increase in number, is achieved ex vivo.

[0187] "Engraftment" or "engraft" refers to the transfer of cells into the body of a recipient, e.g., a mammalian or human subject. In one example, engraftment refers to the uptake of a population of cells or tissue, e.g., HSPCs, into the recipient. In one example, engraftment of HSPCs includes growth, proliferation, and / or differentiation of the engrafted cells in the host. The differentiation and growth of the HSPCs into erythroid cells within the recipient's body.

[0188] The term "hematopoietic progenitor cells" (HPCs), as used herein, refers to cells with limited self-renewal capacity. and multilineage differentiation (e.g., myeloid, lymphoid) depending on their position within the hematopoietic hierarchy. , unilineage differentiation (e.g., myeloid or lymphoid) or cell type restricted differentiation (e.g., erythroid progenitors) These cells are primitive hematopoietic cells with the potential to produce hematopoietic stem cells (Doulatov et al. ,Cell Stem Cell 2012).

[0189] "Hematopoietic stem cells" (HSCs), as used herein, are cells that self-renew and give rise to granulocytes (e.g. promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, erythrocytes), Thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages, These cells are immature blood cells that have the ability to differentiate into more mature blood cells, including erythrocytes (erythrophages). Throughout this specification, HSCs are referred to synonymously as stem cells. It is known that such cells may or may not contain CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. The cells are believed to represent a subpopulation of cells that possess stem cell properties as defined above. C may be derived from primitive progenitor cells (e.g., multipotent progenitor cells) and / or specific hematopoietic lineages (e.g., are pluripotent cells that can give rise to committed progenitor cells (lymphoid progenitor cells) It is well known in the art that stem cells committed to specific hematopoietic lineages are T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and / or lymphoid tissue They can be of a specific macrophage cell lineage. Additionally, HSCs can be long-term HSCs (LT It also refers to short-term HSC (ST-HSC) and short-term HSC (LT-HSC). However, LT-HSCs have unlimited self-renewal potential. (i.e., they survive throughout adulthood), whereas ST-HSCs have limited self-renewal. (i.e., it is only viable for a limited period of time). Any of these HSCs can be used in any of the methods. HSCs are an optional choice because they are highly proliferative and therefore rapidly expand in number. ST-HSCs are useful in this context. Hematopoietic stem cells are optionally obtained from blood products. Liquid preparations include preparations obtained from the body or body organs that contain cells of hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, peripheral blood (e.g., mobilized peripheral blood, e.g., G-CSF) or mobilized with a mobilizing agent such as Plerixafor® (AMD3100) or a combination of G-CSF and Plerixafor® (AMD3100) (See above), liver, thymus, lymph, and spleen. All of the above crude or unfractionated blood products can be enriched for cells with hematopoietic stem cell characteristics by methods known to those skilled in the art. In one embodiment, the HSCs are CD34+ / CD38- / CD90+ / CD45RA In an embodiment, the HSCs are characterized as CD34+ / CD90+ / CD In embodiments, HSCs are characterized as CD34+ cells. In embodiments, HSCs are characterized as CD34+ / CD90+ cells. In an embodiment, HSCs are characterized as CD34+ / CD90+ / CD45RA- characterized as cells.

[0190] "Proliferation" or "proliferating" in relation to cells may or may not be the same. Proliferation refers to an increase in the number of one or more characteristic cell types from an initial population of cells. The initial cells used need not be the same as the cells resulting from the expansion.

[0191] A "cell population" is a cell population isolated from a biological source, such as a blood product or tissue, and It refers to eukaryotic mammalian, preferably human, cells derived from the above cells.

[0192] "Enriched," when used in reference to a cell population, refers to a cell that is enriched for one or more markers, For example, it refers to a cell population selected based on the presence of CD34+.

[0193] The term "CD34+ cells" refers to cells that express the CD34 marker on their surface. CD34+ cells can be detected and analyzed using, for example, flow cytometry and fluorescently labeled anti-CD34 antibodies. and can be counted.

[0194] "Enriched for CD34+ cells" means that the cell population was enriched based on the presence of the CD34 marker. Therefore, the CD34+ cells in the cell population after the selection method The percentage of C cells in the initial cell population before the selection step based on the CD34 marker The percentage of CD34+ cells is higher than that of CD34+ cells. At least 50%, 60%, 70%, 80% or less of the cells in the enriched cell population It could account for at least 90%.

[0195] The terms "F cells" and "F-cells" refer to cells that contain and / or produce fetal hemoglobin (e.g., It refers to cells, usually erythrocytes (e.g., red blood cells), e.g., F-cells are cells that contain or produce detectable levels of fetal hemoglobin. For example, F- The cells are cells that contain or produce at least 5 picograms of fetal hemoglobin. In some embodiments, the F-cells contain or produce at least 6 picograms of fetal hemoglobin. In another example, F-cells are cells that express at least 7 picograms of fetal hemoglobin. In another example, F-cells are cells that contain or produce at least 8 picograms. In another example, F-cells are cells that contain or produce lamb fetal hemoglobin. In another example, the cells contain or produce at least 9 picograms of fetal hemoglobin. In this context, F-cells are cells that contain or produce at least 10 picograms of fetal hemoglobin. Fetal hemoglobin levels can be determined using the assays described herein or by Other methods known in the art, such as fetal hemoglobin detection reagents, can also be used. flow cytometry, high-performance liquid chromatography, mass spectrometry, or enzyme-linked immunosorbent assay It can be measured by an assay.

[0196] Unless otherwise specified, all genomic or chromosomal coordinates are based on hg38.

[0197] The gRNA molecules, compositions and methods described herein are directed to CRISPR / Cas9 systems. The present invention relates to genome editing in eukaryotic cells using the gene encoding the genomic DNA described herein. RNA molecules, compositions and methods relate to regulating globin levels, e.g., globin genes The gRNA molecules, compositions and methods are useful in regulating the expression and production of genes and proteins. The methods may be useful in the treatment of hemoglobinopathies.

[0198] I.gRNA molecule A gRNA molecule may have any number of domains, as described in more detail below. However, gRNA molecules typically contain at least a crRNA domain (a targeting domain). It contains a gene and a tracr gene. It is used as a component of the CRISPR system. The gRNA molecules of the present invention may be used to modify DNA at or near the target site (e.g., Such modifications include, for example, the expression of a functional product of a gene containing the target site. These and further uses include deletions and / or insertions that result in a reduction or elimination of The use is explained in more detail below.

[0199] In one embodiment, the single molecule, i.e., sgRNA, preferably has a 5' to 3' sequence, rRNA (a region that forms part of the targeting domain and flagpole that is complementary to the target sequence) (i.e., containing the crRNA flagpole region); loop; and tracr(c A domain complementary to the rRNA flagpole region, and a nuclease or other effector (containing a domain that further binds a molecule, e.g., a Cas molecule, e.g., a Cas9 molecule) and can take the following formats (5' to 3'): [targeting domain]-[crRNA flagpole region]-[optional first flagpole region] pole extension]-[loop]-[optional first tracr extension]-[tracr ragpole region]-[tracr nuclease binding domain].

[0200] In embodiments, the tracr nuclease binding domain is linked to a Cas protein, e.g. Binds to the Cas9 protein.

[0201] In one embodiment, a bimolecular molecule, i.e., a dgRNA, comprises two polynucleotides: a first a polynucleotide, preferably from 5' to 3', comprising a crRNA (a target sequence complementary to the target sequence) a region forming part of the flagpole and a domain; and a second polynucleotide. tide, preferably 5' to 3', complementary to the tracr (crRNA flagpole region) domains, and nucleases or other effector molecules, such as Cas molecules, e.g., C and a domain that further binds the as9 molecule, and may take the following format: (5' to 3'): Polynucleotide 1 (crRNA):[targeting domain]-[crRNA flagpole Area]-[Optional First Flagpole Extension]-[Optional Second Flagpole extension part] Polynucleotide 2 (tracr): [optional first tracr extension]-[tra cr flagpole region]-[tracr nuclease binding domain].

[0202] In embodiments, the tracr nuclease binding domain is linked to a Cas protein, e.g. Binds to the Cas9 protein.

[0203] In some embodiments, the targeting domain comprises a targeting domain sequence described herein: For example, a targeting domain listed in Table 1, or a targeting domain sequence listed in Table 1 or comprising 17, 18, 19 or 20 (preferably 20) consecutive nucleotides; It comprises or consists of a targeting domain consisting thereof.

[0204] In some embodiments, the flagpole, e.g., the crRNA flagpole region, is located at the 5' 3' to contains GUUUUAGAGCUA (SEQ ID NO: 182).

[0205] In some embodiments, the flagpole, e.g., the crRNA flagpole region, is located at the 5' 3' to contains GUUUAAGAGCUA (SEQ ID NO: 183).

[0206] In some embodiments, the loop comprises, 5' to 3', GAAA (SEQ ID NO: 186). .

[0207] In some embodiments, tracr is, from 5' to 3', [ka] and preferably used in a gRNA molecule comprising SEQ ID NO: 182.

[0208] In some embodiments, tracr is, from 5' to 3', [ka] and preferably used in a gRNA molecule comprising SEQ ID NO: 183.

[0209] In some embodiments, the gRNA may also include an additional U nucleic acid at the 3' end. The RNA may contain an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 U nuclei at the 3' end. In one embodiment, the gRNA may contain an additional nucleotide at the 3' end. In the case of a dgRNA, the polynucleotide of the dgRNA (e.g., the target a polynucleotide containing a targeting domain and a polynucleotide containing a tracr For example, in the case of dgRNA, the 3′ end of the dgRNA may contain an additional U nucleic acid. Polynucleotides (e.g., polynucleotides containing a targeting domain and tracr polynucleotide) at least one of which has an additional 1, 2, 3, 4, 5, 6, 7, 8, In one embodiment, the dgR may comprise 9 or 10 U nucleic acids (SEQ ID NO: 249). In the case of NA, a polynucleotide of the dgRNA (e.g., a polynucleotide containing a targeting domain) and tracr) at least one of which has an additional four In a dgRNA embodiment, only the polynucleotide containing the tracr In some embodiments of the dgRNA, the targeting Only the polynucleotide containing the domain contains an additional U nucleic acid. A polynucleotide comprising a targeting domain and a polynucleotide comprising tracr Both of the amino acids contain an additional U nucleic acid, for example, four U nucleic acids.

[0210] In some embodiments, the gRNA may also include an additional A nucleic acid at the 3' end. The RNA may contain an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 A nuclei at the 3' end. In one embodiment, the gRNA may contain an additional nucleotide at the 3' end. In the case of a dgRNA, the polynucleotide of the dgRNA (e.g., the target a polynucleotide containing a targeting domain and a polynucleotide containing a tracr For example, in the case of dgRNA, the 3' end of the dgRNA may contain an additional A nucleic acid. Polynucleotides (e.g., polynucleotides containing a targeting domain and tracr polynucleotide) at least one of which has an additional 1, 2, 3, 4, 5, 6, 7, 8, In one embodiment, the dgR may comprise 9 or 10 A nucleic acids (SEQ ID NO: 250). In the case of NA, a polynucleotide of the dgRNA (e.g., a polynucleotide containing a targeting domain) and tracr) at least one of which has an additional four In a dgRNA embodiment, only the polynucleotide comprising tracr In some embodiments of the dgRNA, the targeting Only the polynucleotide containing the domain contains an additional A nucleic acid. A polynucleotide comprising a targeting domain and a polynucleotide comprising tracr Both of the amino acids contain an additional U nucleic acid, for example, four A nucleic acids.

[0211] In embodiments, one or more of the polynucleotides of the gRNA molecule are capped at the 5' end. may include:

[0212] In one embodiment, the single molecule, i.e., sgRNA, is preferably , crRNA (containing a targeting domain complementary to the target sequence; crRNA flag domain) loop region; first flagpole extension; loop; first tracr extension (first flagpole containing a domain complementary to at least a portion of the nucleotide extension; and tracr( A domain complementary to the crRNA flagpole region and a Cas9 molecule are further attached. In some embodiments, the targeting domain comprises a targeting domain described herein. A targeting domain sequence that can be used, for example, a targeting domain described in Table 1, or a targeting domain described in Table 1 17, 18, 19, or 20 (preferably 20) consecutive targeting domain sequences nucleotides 3' to the targeting domain sequence listed in Table 1, e.g., nucleotides 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, Containing or consisting of 19 or 20 (preferably 20) consecutive nucleotides Contains a targeting domain.

[0213] In embodiments including a first flagpole extension and / or a first tracr extension, The flagpole, loop and tracr sequences can be as described above. Any first flagpole extension and first tracr extension may be used, but In an embodiment, the first flagpole extension and and the first tracr extension may have 3, 4, 5, 6, 7, 8, 9, 10 or more phases. It consists of complementary nucleotides.

[0214] In some embodiments, the first flagpole extension comprises, from 5' to 3', UGCUG( In some embodiments, the first flagpole extension comprises SEQ ID NO: 184. It consists of No. 184.

[0215] In some embodiments, the first tracr extension comprises, from 5' to 3', CAGCA (sequence In some embodiments, the first tracr extension comprises SEQ ID NO: 189. It consists of:

[0216] In some embodiments, the dgRNA comprises two nucleic acid molecules. The gRNA preferably comprises, from 5' to 3', a targeting domain complementary to the target sequence; A flagpole region; optionally a first flagpole extension; and optionally a second flagpole extension. a first nucleic acid containing a flagpole extension; preferably 5' to 3', optionally The first tracr extension; and the tracr (a domain complementary to the crRNA flagpole region) a domain that further binds a Cas, e.g., Cas9 molecule) a second nucleic acid (which may be referred to herein as tracr), and at least a Cas molecule, e.g. The second nucleic acid further comprises a 3'-terminal domain (e.g., a domain that binds to a Cas9 molecule). It may contain additional U nucleic acids at the ends (e.g., 3' to tracr). , which has additional 1, 2, 3, 4, 5, 6, 7 at the 3' end (e.g., 3' side of tracr) The second nucleic acid may additionally comprise 8, 9, or 10 U nucleic acids (SEQ ID NO: 249). may instead include an additional A nucleic acid at the 3' end (eg, 3' to tracr). For example, tracr may contain an additional 1, 2, or 3′ site at the 3′ end (e.g., on the 3′ side of tracr). It may contain 3, 4, 5, 6, 7, 8, 9, or 10 A nucleic acids (SEQ ID NO: 250). In embodiments, the targeting domain comprises a targeting domain sequence described herein, e.g., , a targeting domain listed in Table 1, or 17 of the targeting domain sequences listed in Table 1, or containing 18, 19 or 20 (preferably 20) consecutive nucleotides. The targeting domain comprises:

[0217] In embodiments involving dgRNA, the crRNA flagpole region, optional first The flagpole extension, the optional first tracr extension, and the tracr sequence are as described above. It can be as described.

[0218] In some embodiments, the optional second flagpole extension comprises, from 5' to 3', a U Contains UUUG (SEQ ID NO: 185).

[0219] In embodiments, gRNA molecules (and in the case of dgRNA molecules, include a targeting domain) 1, 2, 3 on the 3' side of the polynucleotide and / or polynucleotide containing tracr , 4, or 5 nucleotides, 1, 2, 3, 4, or 5 nucleotides on the 5' side, or 3' side and 5' 1, 2, 3, 4, or 5 nucleotides are as described in Section XIII below. As explained in detail in, it is a modified nucleic acid.

[0220] These domains are briefly discussed below: 1) Targeted domains: For guidance on targeting domain selection, see, e.g., Fu Y el al.NA T BIOTECHNOL 2014(doi:10.1038 / nbt.2808) and and Sternberg SH el al.NATURE 2014(doi:10.1 038 / naturel3011).

[0221] The targeting domain is complementary to, e.g., at least 80, 85, 9, or 100 amino acids of, a target sequence on a target nucleic acid. The targeting domain may comprise a nucleotide sequence that is 0, 95, or 99% complementary, e.g., completely complementary. The base is part of an RNA molecule and therefore contains the base uracil (U). On the other hand, any DNA encoding a gRNA molecule will contain the base thymine (T). While not wishing to be bound by this, the complementarity of the targeting domain with the target sequence is This is thought to contribute to the specificity of the interaction of the gRNA / Cas9 complex with the target nucleic acid. In the pair of the targeting domain and the target sequence, the uracil base in the targeting domain is It is understood that it can pair with an adenine base in the target sequence.

[0222] In one embodiment, the targeting domain comprises 5 to 50, for example 10 to 40, for example 10 In one embodiment, the length is from 15 to 30, for example from 15 to 30, for example from 15 to 25 nucleotides. The targeting domains are 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24. In one embodiment, the targeting domain is 16 nucleotides in length. In one embodiment, the targeting domain is 17 nucleotides in length. In one embodiment, the targeting domain is 18 nucleotides in length. In one embodiment, the targeting domain is 19 nucleotides in length. In embodiments, the targeting domain is 20 nucleotides in length. 8, 19, or 20 nucleotides from the targeting domain listed in Table 1, In embodiments, the aforementioned 16, 17, 18, 19, or 20 nucleotides are included. 18, 19, or 20 nucleotides from the 3'-16 from the targeting domain described in Table 1 , 17, 18, 19 or 20 nucleotides.

[0223] Without being bound by theory, the target domain may be located at the 3' end of the targeting domain. 8, 9, 10, 11, or 12 nucleic acids of the target domain are important for targeting the target sequence. and therefore may be referred to as the "core" region of the targeting domain. In the present invention, the core domain is perfectly complementary to the target sequence.

[0224] The strand of the target nucleic acid to which the targeting domain is complementary is referred to herein as the target sequence. In some embodiments, the target sequence is located on a chromosome, e.g., is an intragenic target. In some embodiments, the target sequence is located within an exon of the gene. The sequence is located within an intron of the gene. or containing binding sites for other regulatory elements, such as promoters or transcription factor binding sites. Its proximity (e.g., 10, 20, 30, 40, 50, 100, 200, 300 , 400, 500, or 1000 nucleotides). All may have modifications such as those listed in Section XIII herein.

[0225] 2) crRNA flagpole region: The flagpole contains portions from both crRNA and tracr. The flagpole region is complementary to a portion of tracr, and in one embodiment, at least Under some physiological conditions, e.g., normal physiological conditions, the In one embodiment, the crRNA flag sequence is complementary to a portion of the tracr sequence. In one embodiment, the crRNA flag region is 5 to 30 nucleotides in length. The flagpole region is 5–25 nucleotides in length. The crRNA flagpole region is expressed in the bacterial C share homology with a naturally occurring portion of the repeat sequence from the RISPR array, or In one embodiment, this can be derived from the crRNA fragment disclosed herein. Streptococcus pyogenes (S. pyogenes) or S. thermophilus S. thermophilus crRNA flagpole region and at least 50% It has the following homology.

[0226] In one embodiment, the flagpole, e.g., the crRNA flagpole region, is In one embodiment, a flagpole, e.g., a crRNA flagpole, The fragment region has at least 50%, 60%, 70%, 80%, 85% homology with SEQ ID NO: 182. In one embodiment, the fragment comprises a sequence having 90%, 95%, or 99% homology thereto. The flagpole, e.g., the crRNA flagpole region, comprises at least 5 of SEQ ID NO: 182, In some embodiments, the flag sequence comprises 6, 7, 8, 9, 10, or 11 nucleotides. The pole, e.g., the crRNA flagpole region, comprises SEQ ID NO: 183. In the flagpole, the flagpole is at least 50%, 60%, 70%, 80% or more of the In some embodiments, the sequence may have 0%, 85%, 90%, 95% or 99% homology. In this case, the flagpole, e.g., the crRNA flagpole region, is a small fragment of SEQ ID NO: 183. It contains at least 5, 6, 7, 8, 9, 10, or 11 nucleotides.

[0227] Some or all of the nucleotides of the domain may be modified, for example, as described in Section XIII herein. The amino acid sequence may have the modifications described above.

[0228] 3) First flagpole extension When a tracr containing the first tracr extension is used, the crRNA is Generally, any first flagpole extension and first tr acr extensions can be used, provided they are complementary. The first flagpole extension and the first tracr extension are 3, 4, 5, 6, 7 , 8, 9, 10 or more complementary nucleotides.

[0229] The first flagpole extension is complementary to the nucleotides of the first tracr extension, e.g., For example, 80%, 85%, 90%, 95% or 99%, e.g., fully complementary nucleotides. In some embodiments, the complementary nucleotide of the first tracr extension may be hybridized to the The nucleotides of the first flagpole extension that are rearranged are contiguous. a first flash nucleotide that hybridizes to the complementary nucleotide of the first tracr extension; The nucleotides of the first tracr extension are discontinuous, e.g., Two or more hybridizations separated by nucleotides that do not base pair with the nucleotide In some embodiments, the first flagpole extension comprises at least two , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 In some embodiments, the first fragment comprises 8, 19, 20 or more nucleotides. The tag pole extension comprises, from 5' to 3', UGCUG (SEQ ID NO: 184). In some embodiments, the first flagpole extension consists of SEQ ID NO: 184. The first flagpole extension is at least 80%, 85%, 90% or more of SEQ ID NO: 184. %, 95% or 99% homologous to the nucleic acid.

[0230] Some or all of the nucleotides of the first tracr extension may be modified, e.g., as described herein in It may have the modifications listed in Section XIII.

[0231] 3) Loop The loop is located between the crRNA flagpole region (or optional region, if present) of the sgRNA. tracr (or optionally the first flagpole extension, if present) The loop serves to connect with the crRNA flagpole region. and tracr can be covalently or non-covalently linked. In one embodiment, the loop is linked to the crRNA flagpole. In one embodiment, the loop is Covalently coupling the first flagpole extension to the first tracr extension In one embodiment, the loop comprises a crRNA flagpole region and a crRNA flagpole region. Covalent bond between the tracr domain that hybridizes to the RagPole region Typically, the loop is one or more nucleotides, e.g., 2, 3 , 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0232] In a dgRNA molecule, the two molecules are linked by at least a portion of the crRNA (e.g., crR NA flagpole region) and at least a portion of the tracr (e.g., crRNA flagpole region) by hybridization between the tracr domain (complementary to the globular region of the tracr can meet.

[0233] A variety of loops are suitable for use in sgRNAs. The loop can consist of a covalent bond, or as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length In certain embodiments, the loop may be 2, 3, 4, 5, 6, 7, 8, 9, 10 , 15, 20, or 25 nucleotides in length or more. The loop may be 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides. In one embodiment, the loop shares homology with a naturally occurring sequence. In certain embodiments, the loop is formed from or derived from a loop as disclosed herein. In one embodiment, the loop has at least 50% homology to SEQ ID NO: 1. Including 86.

[0234] Some or all of the nucleotides of the domain may be modified, for example, as described in Section XIII herein. The amino acid sequence may have the modifications described above.

[0235] 4) Second flagpole extension In one embodiment, the dgRNA is a fragment of the crRNA flagpole region or a fragment thereof. In this case, the second flagpole is attached to the 3' side of the first flagpole extension. In one embodiment, the second flagpole may contain an additional sequence called a flagpole extension. The chain extension may be 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides. In some embodiments, the second flagpole extension is 2, 3, 4, It is 5, 6, 7, 8, 9, or 10 nucleotides in length or more. wherein the second flagpole extension comprises SEQ ID NO: 185.

[0236] 5)Tracr: tracr is a nucleic acid sequence required for the binding of a nuclease, such as Cas9. Although not limited thereto, each Cas9 species is associated with a specific tracr sequence. The tracr sequence is used in both sgRNA and dgRNA systems. In one embodiment, tracr is used to treat Streptococcus pyogenes (S. pyogenes) In some embodiments, tracr comprises or is derived from a sequence from tracr. , at least a portion that hybridizes to the flagpole portion of the crRNA, e.g., The crRNA flagpole region is sufficient to form a double-stranded region under physiological conditions. The complementary portion (sometimes referred to herein as the tracr flagpole region or cr It contains a tracr domain (sometimes called a tracr domain) that is complementary to the flagpole region of the RNA. In an embodiment, a tracr that hybridizes with the crRNA flagpole region is The domain hybridizes with the complementary nucleotides of the crRNA flagpole region. At least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 In some embodiments, the crRNA flagpole comprises 8, 19, or 20 nucleotides. The tracr nucleotides that hybridize to the complementary nucleotides of the region are contiguous. In some embodiments, the complementary nucleotides of the crRNA flagpole region are hybridized to the The corresponding tracr nucleotides are discontinuous, e.g., in the crRNA flagpole region. Two or more hybrids separated by nucleotides that do not base pair with the nucleotides in In some embodiments, the crRNA flagpole region contains a hybridization region. The portion of the tracr that is resized is UAGCAAGUUAAAA (sequence number) from 5' to 3'. In some embodiments, the fragments hybridizing to the crRNA flagpole region include: The soybean tracr portion is located 5' to 3' and contains UAGCAAGUUUAAA (sequence In an embodiment, the fragment hybridizes to the crRNA flagpole region. The encoding sequence further binds a nuclease, e.g., a Cas molecule, e.g., a Cas9 molecule. It is located on the tracr 5' side of the tracr sequence.

[0237] The tracr further binds to a nuclease, e.g., a Cas molecule, e.g., a Cas9 molecule. Without being bound by theory, different species of Cas9 are believed to bind to different tracr sequences. In some embodiments, tracr is Contains a sequence that binds to the S. pyogenes Cas9 molecule. In a similar manner, tracr comprises a sequence that binds to a Cas9 molecule disclosed herein. In some embodiments, the domain further attaching the Cas9 molecule comprises, 5' to 3': UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGC (SEQ ID NO: 193) In some embodiments, the domain further attaching the Cas9 molecule comprises a 5' to 3' To, UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 194) Includes:

[0238] In some embodiments, tracr comprises SEQ ID NO: 187. and tracr comprises SEQ ID NO: 188.

[0239] Some or all of the nucleotides of tracr may be modified, e.g., as described in Section XIII herein. In embodiments, the gRNA (e.g., sgRNA or dgRNA) may have the modifications listed. RNA tracr and / or crRNA), e.g., gRNA or gRNA described above Any of the RNA components may be located at the 5' end, the 3' end, or both the 5' and 3' ends of the gRNA. In embodiments, the gRNA (e.g., sgRNA or dgRNA) contains an inverted abasic residue. RNA tracr and / or crRNA), e.g., gRNA or gRNA described above Any of the RNA components may have one or more phosphorothioates between the residues at the 5' end of the polynucleotide. containing a hydroxylate bond, e.g., between the first two 5' residues, between each of the first three 5' residues, between each of the first four 5' residues, or between each of the first five 5' residues In embodiments, the gRNA or gRNA component may instead contain a thioate bond. or in addition, one or more phosphorothioate bonds between residues at the 3' end of the polynucleotide For example, between the first two 3' residues, between each of the first three 3' residues, between the first four phosphorothioate bonds between each of the first three 3' residues or between each of the first five 3' residues In some embodiments, the gRNA (e.g., sgRNA or dgRNA) tracr and / or crRNA), e.g., gRNA or gRNA components described above. One of the fragments contains a phosphorothioate bond between each of the first four 5' residues (e.g., For example, the 5' end comprises, for example, three phosphorothioate bonds, and containing a phosphorothioate bond between each of the first four 3' residues (e.g., 3' at the 3' end) In one embodiment, the above Any of the phosphorothioate modifications described above may be present at the 5' end, 3' end, or 4' end of the polynucleotide. The nucleotides are combined with inverted abasic residues at the 5' or 3' termini. In some embodiments, the inverted abasic nucleotide is a phosphate or phosphorothioate linkage. In an embodiment, the gRNA may be linked to the 5' and / or 3' nucleotide by (e.g., tracr and / or crRNA of sgRNA or dgRNA), e.g., Any of the gRNAs or gRNA components described above may contain nucleotides containing 2'O-methyl modifications. In embodiments, the first one, two, three or all of the 5' residues are In an embodiment, each of the above contains a 2' O-methyl modification. , two, three or more of which each contain a 2'O-methyl modification. , the fourth, third and second penultimate 3' residues are 2'O-methyl modified In embodiments, each of the first one, two, three or more 5' residues comprises: contains 2'O-methyl modifications and the first one, two, three or more of the 3' residues Each of the first three 5' residues contains a 2'O-methyl modification. each containing a 2'O-methyl modification, and each of the first three 3' residues is a 2'O-methyl modification In an embodiment, each of the first three 5' residues comprises a 2'O-methyl modification, and the 4th, 3rd and 2nd penultimate 3' residues are 2'O-methyl modified. In embodiments, any of the 2'O-methyl modifications, such as those described above, may be used. and wherein the nucleic acid has one or more phosphorothioate modifications, e.g., as described above. and / or one or more inverted abasic modifications, e.g., in combination with those described above. In some embodiments, the tr of a gRNA (e.g., an sgRNA or a dgRNA) acr and / or crRNA), e.g., gRNA or gRNA components described above Either one has a phosphorothioate bond between each of the first four 5' residues (e.g., poly( (e.g., consisting of) three phosphorothioate bonds at the 5' end of the nucleotide , a phosphorothioate bond (e.g., a polynucleotide) between each of the first four 3' residues the first three 2'O-methyl modification at each of the first three 5' residues, and 2'O- In some embodiments, the gRNA (e.g., s gRNA or dgRNA, tracr and / or crRNA), e.g., as described above Either the gRNA or the gRNA component contains a phosphorothioate between each of the first four 5' residues. Thioate linkages (e.g., three phosphorothioate linkages at the 5' end of a polynucleotide) a phosphorothioate residue between each of the first four 3' residues linkages (e.g., containing three phosphorothioate linkages at the 5' end of the polynucleotide, e.g., 2' O-methyl modifications at each of the first three 5' residues, and Contains 2'O-methyl modifications at each of the fourth, third and second-to-last 3' residues Hmm, for example, it consists of that.

[0240] In one embodiment, the trajectory and and / or crRNA), e.g., any of the gRNAs or gRNA components described above is a polynucleotide with a phosphorothioate bond between each of the first four 5' residues (e.g., containing, e.g., consisting of, three phosphorothioate bonds at the 5' end of the Phosphorothioate linkages between each of the four 3' residues (e.g., the 5' the first three 5' 2'O-methyl modifications on each of the residues, 2'O-methyl modifications on each of the first three 3' residues, and comprising, e.g., consisting of, an additional inverted abasic residue at each of the 5' and 3' ends .

[0241] In one embodiment, the trajectory and and / or crRNA), e.g., any of the gRNAs or gRNA components described above is a polynucleotide with a phosphorothioate bond between each of the first four 5' residues (e.g., containing, e.g., consisting of, three phosphorothioate bonds at the 5' end of the Phosphorothioate linkages between each of the four 3' residues (e.g., the 5' the first three 5' Each of the residues was modified with 2'O-methyl, and the penultimate, third, and second 2'O-methyl modification at each of the 3' residues, and additional 5' and 3' residues at each of the 5' and 3' ends. The amino acid sequence may comprise, for example consist of, an inverted abasic residue.

[0242] In one embodiment, the gRNA is a dgRNA and comprises, for example, R: crRNA: mN*mN*mN*NNNNNNNNNNNNNNNNGUUUUAGAGCUAU* mG*mC*mU (SEQ ID NO: 251) (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends; and tracr: [ka] (Optionally with inverted abasic residues at the 5' and / or 3' ends).

[0243] In one embodiment, the gRNA is a dgRNA and comprises, for example, R: crRNA: mN*mN*mN*NNNNNNNNNNNNNNNNGUUUUAGAGCUAU* mG*mC*mU (SEQ ID NO: 251) (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends; and tracr: [ka] (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends).

[0244] In one embodiment, the gRNA is a dgRNA and comprises, for example, R: crRNA: mN*mN*mN*NNNNNNNNNNNNNNNNGUUUUAGAGCUAUG CUGUU*mU*mU*mG (SEQ ID NO: 252) (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends; and tracr: [ka] (Optionally with inverted abasic residues at the 5' and / or 3' ends).

[0245] In one embodiment, the gRNA is a dgRNA and comprises, for example, R: crRNA: mN*mN*mN*NNNNNNNNNNNNNNNNGUUUUAGAGCUAUG CUGUU*mU*mU*mG (SEQ ID NO: 252) (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends; and tracr: [ka] where m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond. ) (optionally with inverted abasic residues at the 5' and / or 3' ends).

[0246] In one embodiment, the gRNA is a dgRNA and comprises, for example, R: crRNA: NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUU UG (SEQ ID NO: 253) (wherein N represents a residue of a targeting domain, e.g., as described herein) (optional) optionally with inverted abasic residues at the 5' and / or 3' ends; and tracr: [ka] where m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond. ) (optionally with inverted abasic residues at the 5' and / or 3' ends).

[0247] In one embodiment, the gRNA is an sgRNA and comprises, for example, R: [ka] (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends).

[0248] In one embodiment, the gRNA is an sgRNA and comprises, for example, R: [ka] (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends).

[0249] In one embodiment, the gRNA is an sgRNA and comprises, for example, R: [ka] (wherein m represents a base having a 2'O-methyl modification, and * represents a phosphorothioate bond.) and N represents a residue of a targeting domain, e.g., as described herein) (optional). optionally with inverted abasic residues at the 5' and / or 3' ends).

[0250] 6) First Tracr extension If the gRNA contains the first flagpole extension, the tracr will be The first tracr extension may comprise a nucleotide sequence of the first flagpole extension. complementary to the nucleotide sequence, e.g., 80%, 85%, 90%, 95%, or 99%, e.g., fully complementary In some embodiments, the complementary nucleotide of the first flagpole extension may be The first tracr extension nucleotides that hybridize to the nucleotide are contiguous. In some embodiments, a complementary nucleotide of the first flagpole extension hybridizes to the complementary nucleotide of the first flagpole extension. The first tracr extension nucleotides are discontinuous, e.g., the first flag Two or more half-strands separated by nucleotides that do not base pair with the nucleotides in the half-strand extension In some embodiments, the first tracr extension comprises a hybridization region. At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 In some embodiments, the amino acid sequence comprises 17, 18, 19, 20 or more nucleotides. The first tracr extension comprises SEQ ID NO: 189. In some embodiments, the first tracr The r extension is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 189. Includes nucleic acids that are homologous.

[0251] Some or all of the nucleotides of the first tracr extension may be modified, e.g., as described herein in It may have the modifications listed in Section XIII.

[0252] In some embodiments, the sgRNA is 5' to 3', 3' to the targeting domain. It may include arranged: [ka] e) at least 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides at the 3' end further comprising, for example, 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides. Any of the above a) to d); f) at least 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides at the 3' end further comprising, for example, 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides. Any of the above a) to d); or g) at least one nucleotide sequence at the 5' end (e.g., at the 5' end, e.g., 5' to the targeting domain), , 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, Any of the above a) to f) further containing 5, 6 or 7 adenine (A) nucleotides. In an embodiment, any of the above a) to g) is located immediately 3' to the targeting domain. will be done.

[0253] In one embodiment, the sgRNA of the invention comprises, from 5' to 3', a [targeting domain] - [ka] comprises, for example consists of,

[0254] In one embodiment, the sgRNA of the invention comprises, from 5' to 3': [Targeted Domain]- [ka] comprises, for example consists of,

[0255] In some embodiments, the dgRNA may comprise: 5' to 3', preferably immediately 3' of the targeting domain, including: RNA: a) GUUUUAGAGCUA (SEQ ID NO: 182); b) GUUUAAGAGCUA (SEQ ID NO: 183); c) GUUUUAGAGCUAUGCUG (SEQ ID NO: 199); d) GUUUAAGAGCUAUGCUG (SEQ ID NO: 200); e) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 201); f) GUUUAAGAGCUAUGCUGUUUUG (SEQ ID NO: 202); or g) GUUUUAGAGCUAUGCU (SEQ ID NO: 226): and tracr containing the following from 5' to 3': [ka] k) at least 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides at the 3' end further comprising, for example, 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides. Any of the above a) to j); l) at least 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides at the 3' end further comprising, for example, 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides. Any of the above a) to j); or m) at least 1, 2, 3, 4, 5, 6, or 7 amino acids at the 5' end (e.g., at the 5' end); adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides Any of the above a) to l) further containing a nucleotide.

[0256] In one embodiment, the sequence of k) above is, for example, a sequence in which a U6 promoter is used for transcription. In one embodiment, the sequence in k) above is, for example, For example, when the HI promoter is used for transcription, it contains the 3' sequence UUUU. In this case, the sequence k) is, for example, the termination signal of the pol-III promoter used. In one embodiment, the sequence of k) above contains a variable number of 3' Us depending on the sequence. 7 promoters, if used, contain variable 3' sequences derived from the DNA template. In an embodiment, the sequence in k) above is a sequence that is prepared by, for example, in vitro transcription of an RNA molecule. In one embodiment, the above The sequence of item k) is, for example, when transcription is driven using a pol-II promoter, Contains a variable 3' sequence derived from the DNA template.

[0257] In one embodiment, the crRNA comprises a targeting domain and a SEQ ID NO: 201, located in the targeting domain (e.g., immediately 3' to the targeting domain) an array comprising, e.g. consisting of, [ka] tracr, which includes, for example, consists of, and tracr, which includes, for example, consists of,

[0258] In one embodiment, the crRNA comprises a targeting domain and a SEQ ID NO: 202, located (e.g., immediately 3' to the targeting domain) an array comprising, e.g. consisting of, [ka] tracr, which includes, for example, consists of, and tracr, which includes, for example, consists of,

[0259] In one embodiment, the crRNA comprises a targeting domain and a located (e.g., immediately 3' to the targeting domain), GUUUUAGA A sequence comprising, for example consisting of, GCUAUGCU (SEQ ID NO: 226); [ka] tracr, which includes, for example, consists of, and tracr, which includes, for example, consists of,

[0260] In one embodiment, the crRNA comprises a targeting domain and a located (e.g., immediately 3' to the targeting domain), GUUUUAGA A sequence comprising, for example consisting of, GCUAUGCU (SEQ ID NO: 226); [ka] tracr, which includes, for example, consists of, and tracr, which includes, for example, consists of,

[0261] In one embodiment, the crRNA comprises a targeting domain and a located (e.g., immediately 3' to the targeting domain), GUUUUAGA A sequence comprising, for example consisting of, GCUAUGCUGUUUUG (SEQ ID NO: 201); [ka] tracr, which includes, for example, consists of, and tracr, which includes, for example, consists of,

[0262] II. gRNA targeting domain directed to the non-deleted HPFH region The following table provides a list of gRNA molecules of the invention and various embodiments of the invention, such as globin It is used to modify the expression of hemoglobin genes, such as the fetal hemoglobin gene or the hemoglobin β gene. The present invention provides a targeting domain directed to the non-deleted HPFH region, which can be used to target the non-deleted HPFH region.

[0263] [Table 1]

[0264] [Table 2]

[0265] [Table 3]

[0266] [Table 4]

[0267] [Table 5]

[0268] Table 2 below shows the results of the synthesis of gRNA molecules in 7 days according to the method described in the Examples. At least one of fetal hemoglobin (e.g., in erythroid cells differentiated from modified HSPCs) The targeting domains that also result in a 17% increase are shown. gRNA molecules comprising the nucleotide sequence are collectively referred to herein as Tier 2 gRNA molecules. .

[0269] [Table 6]

[0270] Tables 3a and 3b below show the sequences that, when included in a gRNA molecule, can be used in the methods described in the Examples. Therefore, at 7 days (e.g., in erythroid cells differentiated from modified HSPCs), fetal hemoglobin The targeting domains that resulted in the greatest increase in gR activity are shown. NA molecules are collectively referred to herein as Tier 1 (e.g., Tirr 1a or Ti er 1b) called gRNA molecule.

[0271] [Table 7]

[0272] [Table 8]

[0273] III. gRNA Design Method Methods for designing gRNAs are described herein, including methods for selecting, designing, and validating target sequences. Exemplary targeting domains are also provided herein. The targeting domains discussed herein The gene can be incorporated into the gRNA described herein.

[0274] For methods of target sequence selection and validation, as well as off-target analysis, see, e.g., Mali el al.,2013 SCIENCE 339(6121):823-826;H su et al,2013 NAT BIOTECHNOL,31(9):827-3 2;Fu et al,2014 NAT BIOTECHNOL,doi:10.10 38 / nbt.2808.PubMed PM ID:24463574;Heigwe r et al,2014 NAT METHODS ll(2):122-3.doi :10.1038 / nmeth.2812.PubMed PMID:24481216 ;Bae el al,2014 BIOINFORMATICS PubMed PM ID:24463181;Xiao A el al,2014 BIOINFORMA Listed in TICS PubMed PMID:24389662.

[0275] Selection of gRNAs within the user's target sequence using software tools, for example Optimize the target sequence, e.g., minimize overall off-target activity across the genome. Off-target activity can be other than cleavage. The tool provides a , for example, using Streptococcus pyogenes (S. pyogenes) Cas9 to generate a specific number (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatched base pairs Contains all off-target sequences across the genome (e.g., NAG PAM or NGG Cleavage at each off-target sequence (one preceding either the PAM) can be identified. Efficiency can be predicted, for example, using an empirically derived weighting scheme. Each capable gRNA is ranked according to its overall predicted off-target cleavage. gRNAs with the highest on-target cleavage and the lowest off-target cleavage are likely to be Other features, such as automated reagent design for CRISPR construction, Primer design for target Surveyor assays and next-generation sequencing Primer design tools for high-throughput detection and quantification of off-target cleavage by ribosomal RNA Candidate gRNA molecules can be selected by methods known in the art or by methods described herein. This can be determined as described in the specifications.

[0276] Software algorithms can be used to generate an initial list of potential gRNA molecules. Cleavage efficiency and specificity do not necessarily reflect predicted values, and gRNA molecules may be Specifically, certain cell lines, e.g., primary human cell lines, e.g., human HSPCs, e.g., human CD3 4+ cells to assess, for example, cleavage efficiency, indel formation, cleavage specificity, and desired These characteristics can be determined by the methods described herein. The assay can be performed using the following methods.

[0277] In an embodiment of the present invention, the targeting domain of GCR-0001 (SEQ ID NO: 1, underlined) A gRNA comprising the unmodified sequence (as shown), e.g., one of the gRNA molecules described below, can be used in For example, the CRIs of the present invention, including embodiments involving two or more of the gRNA molecules described herein, Useful in SPR systems, methods, cells and other aspects and embodiments. [ka]

[0278] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0279] In an embodiment of the present invention, the targeting domain of GCR-0006 (SEQ ID NO: 6, underlined) A gRNA comprising the unmodified sequence (as shown), e.g., one of the gRNA molecules described below, can be used in For example, the CRIs of the present invention, including embodiments involving two or more of the gRNA molecules described herein, Useful in SPR systems, methods, cells and other aspects and embodiments. [ka]

[0280] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0281] In an embodiment of the present invention, the targeting domain of GCR-0008 (SEQ ID NO: 8, underlined) A gRNA comprising the unmodified sequence (as shown), e.g., one of the gRNA molecules described below, can be used in For example, the CRIs of the present invention, including embodiments involving two or more of the gRNA molecules described herein, Useful in SPR systems, methods, cells and other aspects and embodiments. [ka]

[0282] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0283] In an embodiment of the present invention, the targeting domain of GCR-009 (SEQ ID NO: 9, underlined) A gRNA comprising a modified sequence (e.g., a modified unmodified sequence), such as one of the gRNA molecules described below, can be used, e.g., For example, the CRIS of the present invention, including embodiments involving two or more gRNA molecules as described herein, The present invention is useful in PR systems, methods, cells and other aspects and embodiments. [ka]

[0284] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0285] In an embodiment of the present invention, the targeting domain of GCR-0010 (SEQ ID NO: 10, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0286] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0287] In an embodiment of the present invention, the targeting domain of GCR-0011 (SEQ ID NO: 11, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0288] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0289] In an embodiment of the present invention, the targeting domain of GCR-0012 (SEQ ID NO: 12, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0290] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0291] In an embodiment of the present invention, the targeting domain of GCR-0028 (SEQ ID NO: 28, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0292] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0293] In an embodiment of the present invention, the targeting domain of GCR-0034 (SEQ ID NO: 34, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0294] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0295] In an embodiment of the present invention, the targeting domain of GCR-0045 (SEQ ID NO: 45, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0296] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0297] In an embodiment of the present invention, the targeting domain of GCR-0046 (SEQ ID NO: 46, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0298] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0299] In an embodiment of the present invention, the targeting domain of GCR-0047 (SEQ ID NO: 47, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0300] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0301] In an embodiment of the present invention, the targeting domain of GCR-0048 (SEQ ID NO: 48, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0302] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0303] In an embodiment of the present invention, the targeting domain of GCR-0050 (SEQ ID NO: 50, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0304] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0305] In an embodiment of the present invention, the targeting domain of GCR-0051 (SEQ ID NO: 51, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0306] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0307] In an embodiment of the present invention, the targeting domain of GCR-0053 (SEQ ID NO: 53, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0308] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0309] In an embodiment of the present invention, the targeting domain of GCR-0054 (SEQ ID NO: 54, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0310] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0311] In an embodiment of the present invention, the targeting domain of GCR-0058 (SEQ ID NO: 58, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0312] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0313] In an embodiment of the present invention, the targeting domain of GCR-0062 (SEQ ID NO: 62, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0314] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0315] In an embodiment of the present invention, the targeting domain of GCR-0063 (SEQ ID NO: 63, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0316] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0317] In an embodiment of the present invention, the targeting domain of GCR-0067 (SEQ ID NO: 67, underlined) A gRNA comprising the unmodified sequence (unmodified sequence subtracted from the original sequence), such as one of the gRNA molecules described below, For example, the CR sequences of the present invention, including those involving two or more of the gRNA molecules described herein, are Useful in ISPR systems, methods, cells and other aspects and embodiments. [ka]

[0318] In each of the gRNA molecules described above, an "*" indicates a host sequence between adjacent nucleotides. means a holothioate bond, and "mN" (where N=A, G, C, or U) means a 2'- In embodiments, any of the g modified nucleotides described herein may be used. RNA molecules, such as gRNA molecules described above, can be used in, for example, It complexes with the Cas9 molecule to form a ribonucleoprotein complex (RNP). The RNPs described herein are, for example, those described in the methods, cells, and other aspects and embodiments of the invention. It is particularly useful in the form

[0319] IV.Cas molecule Cas9 molecule In a preferred embodiment, the Cas molecule is a Cas9 molecule. The methods and compositions can use Cas9 molecules from a variety of species. Although the (S. pyogenes) Cas9 molecule is the subject of much of the disclosure herein, Cas9 molecules of other species of Cas9 proteins listed herein, Cas9 molecules derived therefrom molecules, or Cas9 molecules based on them, can be used as well. , other Cas9 molecules, e.g., S. thermophilus , Staphylococcus aureus and / or Neisseria meningitidis ( Neisseria meningitidis Cas9 molecules are described herein. Additional Cas9 species can be used in systems, methods, and compositions. Acidovorax avenae, Actinobacillus pluroni Actinobacillus pleuropneumoniae, Actinobacillus succinogenes nes), Actinobacillus suis, Actinomyces sp., Alicycliphilus denito Alicycliphilus denitrificans, amino Aminomonas paucivorans, Cereus Bacillus cereus, Bacillus smithii ithii), Bacillus thuringiensis (Bacillus thuringi ensis), Bacteroides sp., Blastophile Blastopirellula marina, Bradyrhizobium Bradyrhizobium sp., Brevibacillus laterosporus (B revibacillus laterosporus), Campylobacter coli (C ampylobacter coli), Campylobacter jejuni (Campyl obacter jejuni, Campylobacter rad r lad), Candidatus Punic eispirillum), Clostridium cellulolyticum (Clostridi um cellulolyticum), Clostridium perfringens (Clostridium pe rfringens, Corynebacterium acupuncture Corynebacterium accolens, Corynebacterium diphtheriae htheria, Corynebacterium maturshottii (Corynebacter rium matruchotii), Dinorosceobacter shiitake (Dinoros eobacter shibae), Eubacterium doricum (Eubacteri um dolichum), Gamma proteobacteria (gamma proteobacteria cterium, Gluconacetobacter diazotrophicus (Gluconace Haemophilus influenzae (Haem ophilus parainfluenzae), Haemophilus sputum (Hae mophilus sputorum), Helicobacter canadensis (Helico bacter canadensis, Helicobacter cinerea (Helicoba Helicobacter cinaedi, Helicobacter mustelae mustelae), Ilyobacter polytropus (Ilyobacter pol ytropus), Kingella kingae, Lactobacillus Lactobacillus crispatus, Listeria Listeria ivanovii, Listeria monocytogenes (Listeria monocytogenes), Listeriaceae (Listeria ceae bacteria, Methylocystis sp., Methyl Methylosinus trichosporiu m), Mobiluncus mulieris, Neisse Neisseria bacilliformis, Neisseria Neisseria cinerea, Staphylococcus aureus a flavescens), Neisseria lactamica amica), Neisseria sp., Neisseria waswer Castanopsis wadsworthii, Nitrosomonas species osomonas sp.), Parvibacrum lavamentivorans (Parvibac ulum lavamentivorans), Pasteurella multocida (Pasteu rella multocida), Phascolarctobacterium succinatum (Phascolarctobacterium succinatutens), Lal Ralstonia syzygii, Rhodopseudomonas patens Rustris (Rhodopseudomonas palustris), Rhodoblum spp. species (Rhodovulum sp.), Simonsiella muerelli (Simonsiell a muelleri), Sphingomonas sp. , Sporolactobacillus vinea e), Staphylococcus lugdunensis (Staphylococcus lug dunensis), Streptococcus sp. , Subdoligranulum sp., Tistrela Tistrella mobilis, Treponema species ema sp.), or Verminephro bacter eiseniae).

[0320] A Cas9 molecule, as that term is used herein, refers to a gRNA molecule (e.g., , the sequence of the tracr domain) and cooperate with the gRNA molecule to target the sequence and and PAM sequences (e.g., targeting or directing homing to them). It refers to a molecule that can be (or be) labeled.

[0321] In one embodiment, the Cas9 molecule is capable of cleaving a target nucleic acid molecule, as described herein. In one embodiment, an active Cas9 molecule is nickase activity, i.e., the ability to selectively bind one strand of a nucleic acid molecule, e.g., a non-complementary strand or double-stranded nuclease activity, i.e., the ability to cleave complementary strands; the ability to cleave and create a double-strand break, which in one embodiment is the presence of two nickase activities endonuclease activity; exonuclease activity; and helicase activity, i.e. That is, the ability to unwind the helical structure of double-stranded nucleic acids.

[0322] In one embodiment, the enzymatically active Cas9 molecule cleaves both DNA strands to form a bispecific fragment. In some embodiments, the Cas9 molecule only cleaves one strand, e.g., the g The strand to which the RNA hybridizes, or the strand complementary to the strand to which the gRNA hybridizes In one embodiment, the active Cas9 molecule cleaves a nucleotide sequence associated with an HNH-like domain. In one embodiment, the active Cas9 molecule comprises an N-terminal RuvC-like domain. In one embodiment, the active Cas9 molecule comprises cleavage activity associated with an HNH-like domain. It contains cleavage activity associated with the N-terminal RuvC-like domain and cleavage activity associated with the N-terminal RuvC-like domain. In some embodiments, the active Cas9 molecule comprises an active or cleavage-competent HNH-like domain. and an inactive or cleavage-incompetent N-terminal RuvC-like domain. In embodiments, the active Cas9 molecule is an inactive or cleavage-incompetent HNH-like domain and an active or cleavage-competent N-terminal RuvC-like domain.

[0323] In one embodiment, an active Cas9 molecule exhibits the ability to interact with and cleave a target nucleic acid. The effect is PAM sequence dependent. The PAM sequence is a sequence in the target nucleic acid. In some embodiments, In this case, cleavage of the target nucleic acid occurs upstream from the PAM sequence. The molecules can recognize different sequence motifs (e.g., PAM sequences). In S. pyogenes, the active Cas9 molecule has the sequence motif Recognizes NGG and cleaves the target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs upstream from the sequence For example, Mali el ai, SCIENCE 2013;339(6121 ):823-826. In one embodiment, S. thermophilus (S. The active Cas9 molecule of B. thermophilus contains the sequence motifs NGGNG and NNAG AAW (W=A or T) and 1 to 10, e.g., 3 to 5 base pairs upstream of these sequences. This directs cleavage of the core target nucleic acid sequence. See, e.g., Horvath et al., SCIEN CE 2010;327(5962):167-170, and Deveau et al. ,J BACTERIOL 2008;190(4):1390-1400 In one embodiment, an active Cas9 molecule from S. mutans recognizes the sequence motif NGG or NAAR (RA or G), and from this sequence, 1 to 10, For example, it directs cleavage of the core target nucleic acid sequence 3-5 base pairs upstream. See, e.g., Deveau et al. al.,J BACTERIOL 2008;190(4):1 390-1400 Please refer to.

[0324] In one embodiment, the active Cas9 molecule of S. aureus is Recognize the sequence motif NNGRR (R=A or G) and select 1 to 10, e.g., 3 to 5, from the sequence. This induces cleavage of the target nucleic acid sequence upstream of the base pair. See RE, vol. 520, 2015, pp. 186-191. In this study, the active Cas9 molecule of N. meningitidis was identified as It recognizes the NNNNGATT sequence and targets 1 to 10, e.g., 3 to 5 base pairs upstream from the sequence. This leads to cleavage of the amino acid sequence. See, e.g., Hou et al., PNAS EARLY EDIT See ION 2013, 1-6. The ability of Cas9 molecules to recognize PAM sequences is , e.g., Jinek et al., SCIENCE 2012, 337:816 This can be determined using a transformation assay such as that described above.

[0325] Some Cas9 molecules interact with gRNA molecules and, together with the gRNA molecules, form the core target domain. have the ability to home to (e.g., be targeted or localized to) a target nucleic acid It does not have the ability to cleave the nucleotide sequence or to cleave it at an efficient rate. A Cas9 molecule that is free of, or substantially free of, an inactive Cas9 (enzyme) is referred to herein as an inactive Cas9 (enzyme). These may be referred to as functionally inactive Cas9, dead Cas9, or dCas9 molecules. Inactive Cas9 molecules lack or have substantially reduced cleavage activity, e.g., those described herein. 20, 10, 5, 1 or 0.1% of the reference Cas9 molecule as measured by an assay It may have a cleavage activity of less than 1000 kJ / ml.

[0326] For exemplary naturally occurring Cas9 molecules, see Chylinski et al., RNA Biology 2013;10:5,727-737. The as9 molecule contains cluster 1 bacterial families, cluster 2 bacterial families, and cluster Cluster 3 bacterial families, Cluster 4 bacterial families, Cluster 5 bacterial families, Cluster Star 6 bacterial families, Cluster 7 bacterial families, Cluster 8 bacterial families, Cluster 9 bacterial families, Cluster 10 bacterial families, Cluster 11 bacterial families Milly, Cluster 12 bacterial families, Cluster 13 bacterial families, Cluster 1 4 bacterial families, Cluster 1 bacterial families, Cluster 16 bacterial families, Cluster Star 17 bacterial family, Cluster 18 bacterial family, Cluster 19 bacterial family Lee, Cluster 20 bacterial families, Cluster 21 bacterial families, Cluster 22 Bacterial families, Cluster 23 bacterial families, Cluster 24 bacterial families, Cluster Star 25 bacterial family, Cluster 26 bacterial family, Cluster 27 bacterial family Lee, Cluster 28 bacterial families, Cluster 29 bacterial families, Cluster 30 Bacterial families, Cluster 31 bacterial families, Cluster 32 bacterial families, Cluster Star 33 bacterial family, Cluster 34 bacterial family, Cluster 35 bacterial family Lee, Cluster 36 bacterial families, Cluster 37 bacterial families, Cluster 38 Bacterial families, Cluster 39 bacterial families, Cluster 40 bacterial families, Cluster Star 41 bacterial family, Cluster 42 bacterial family, Cluster 43 bacterial family Lee, Cluster 44 bacterial families, Cluster 45 bacterial families, Cluster 46 Bacterial families, Cluster 47 bacterial families, Cluster 48 bacterial families, Cluster Star 49 bacterial family, Cluster 50 bacterial family, Cluster 51 bacterial family Lee, Cluster 52 bacterial families, Cluster 53 bacterial families, Cluster 54 Bacterial families, Cluster 55 bacterial families, Cluster 56 bacterial families, Cluster Star 57 bacterial family, Cluster 58 bacterial family, Cluster 59 bacterial family Lee, Cluster 60 bacterial families, Cluster 61 bacterial families, Cluster 62 Bacterial families, Cluster 63 bacterial families, Cluster 64 bacterial families, Cluster Star 65 bacterial family, Cluster 66 bacterial family, Cluster 67 bacterial family Lee, Cluster 68 bacterial families, Cluster 69 bacterial families, Cluster 70 Bacterial families, Cluster 71 bacterial families, Cluster 72 bacterial families, Cluster Star 73 bacterial family, Cluster 74 bacterial family, Cluster 75 bacterial family Lee, Cluster 76 bacterial family, Cluster 77 bacterial family, or Cluster Cas9 molecules from 78 bacterial families are included.

[0327] Exemplary naturally occurring Cas9 molecules include Cas9 molecules from cluster 1 bacterial families. Examples include Streptococcus pyogenes (S. pyogenes) (e.g., strain SF 370, MGAS 10270, MGAS 10750, MGAS2096, MGAS3 15, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI -1), S. thermophilus (e.g., strain LMD-9 ), S. pseudoporcinus (e.g., strain SPI N 20026), S. mutans (e.g., strain UA 159, NN2025), S. macacae (e.g., strain NCTC1 155 8), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equinus (e.g., strain A TCC 9812, MGCS 124), S. dysgalactiae (S. dysgal actiae (e.g., strain GGS 124), S. bovis (e.g., , strain ATCC 700338), S. anginosus (e.g. , strain F0211), S. agalactiae (e.g., strain N EM316, A909), Listeria monocytogenes cytogenes (e.g., strain F6854), Listeria innocua (Liste ria innocua) (L. innocua, e.g., strain Cli pl 262), Enterococcus italicus licus) (e.g., strain DSM 15952), or Enterococcus faecium ( Enterococcus faecium) (e.g., strain 1,231,408) Ca Further exemplary Cas9 molecules include the s9 molecule. meningitidis Cas9 molecule (Hou et al. PNAS Ear ly Edition 2013, 1-6) and Staphylococcus aureus (S. aureus) C It is an as9 molecule.

[0328] In one embodiment, a Cas9 molecule, e.g., an active Cas9 molecule or an inactive Cas9 molecule, The molecule may be any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence. sequences, e.g., those listed herein or in Chylinski et al., RNA Biology 2013,10:5,'I2'I-Τ,1,Hou et al.PN Cas9 molecules of species described in AS Early Edition 2013, 1-6 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous amino acid sequence, and 1% or less when compared thereto. Less than 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the amino acid residues differ or 1, 2, 5, 10 or 20 or more amino acids 100, 80, 70, 60, 50, 4 It may differ by 0 or by no more than 30 amino acids, or may be identical thereto.

[0329] In one embodiment, the Cas9 molecule is a Streptococcus pyogenes (S. pyogenes) Cas9: [ka] [ka] [ka] [ka] [ka] and 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, and 97% , 98%, or 99% homologous amino acid sequence, and , 2%, 5%, 10%, 15%, 20%, 30%, or 40% or less of the amino acid residues differ. or 1, 2, 5, 10 or 20 or more amino acids, 100, 80, 70, 60, 50, It may differ by no more than 40 or 30 amino acids, or may be identical thereto.

[0330] In embodiments, the Cas9 molecule is cleaved with a positively charged amino acid (e.g., lysine, arginine, or By introducing an uncharged or nonpolar amino acid, such as alanine, into the position (e.g., histidine), Streptococcus pyogenes (S. pyogenes) of SEQ ID NO: 205 containing one or more mutations In embodiments, the mutation is in one or more nt grooves of Cas9. In embodiments, the Cas9 molecule is a mutation to a positively charged amino acid in the sequence A mutation at position 855 of SEQ ID NO: 205, e.g., a non- Streptococcus pyogenes (S) of SEQ ID NO: 205 containing a mutation to a charged amino acid, e.g., alanine. In embodiments, the Cas9 molecule is a Cas9 mutant of the sequence Only at position 855 of SEQ ID NO: 205 compared to SEQ ID NO: 205, e.g., an uncharged amino acid, e.g., In embodiments, the Cas9 molecule has a mutation to alanine. Mutation at position 810, mutation at position 1003, and / or mutation at position 1060 mutations at positions 810, 1003, and / or 1060 of SEQ ID NO: 205, S. pyogenes (S. pyogenes) of SEQ ID NO: 205 containing a mutation to alanine in s) Cas9 mutants. In embodiments, the Cas9 molecule is and has mutations only at positions 810, 1003, and 1060 of SEQ ID NO: 205, e.g. For example, where each mutation is to an uncharged amino acid, e.g., alanine. In one embodiment, the Cas9 molecule comprises a mutation at position 848 of SEQ ID NO: 205, a mutation at position 1003 ... and / or a mutation at position 1060, e.g., a mutation at position 1061 of SEQ ID NO: 205. Sequences containing mutations to alanine at positions 848, 1003, and / or 1060 Streptococcus pyogenes (S. pyogenes) Cas9 mutant no. 205. In the Cas9 molecule, the amino acid sequence at positions 848, 100, and 110 of SEQ ID NO: 205 is 3, and 1060, where each mutation is an uncharged amino acid. In embodiments, the Cas9 molecule is a mutation to S1 aymaker et al., Science Express (December 1, 2015) Science DOI:10.1126 / science.aad5227 The Cas9 molecule is as described in (available online at http: / / www.cas9.org / ).

[0331] In embodiments, the Cas9 molecule comprises the nucleotide sequence of SEQ ID NO: 205 comprising one or more mutations. In some embodiments, the Cas9 mutant is a Streptococcus pyogenes (S. pyogenes) Cas9 mutant. 9 variants contain a mutation at position 80 of SEQ ID NO:205, e.g., position 80 of SEQ ID NO:205 205, which contains a leucine (i.e., a C80L mutation), e.g., In embodiments, the Cas9 mutant comprises a mutation at position 574 of SEQ ID NO: 205. For example, a glutamic acid at position 574 of SEQ ID NO: 205 (i.e., C574 In an embodiment, the nucleotide sequence of the present invention comprises, for example, consists of, SEQ ID NO: 205 having an E mutation. The Cas9 mutant contains a mutation at position 80 and a mutation at position 574 of SEQ ID NO:205. Mutations include, for example, a leucine at position 80 of SEQ ID NO:205 and a nucleotide at position 574 of SEQ ID NO:205. The sequence containing glutamic acid at position 1 (i.e., the C80L mutation and the C574E mutation) (e.g., consisting of, including column number 205). Without being bound by theory, Such mutations are believed to improve the lytic properties of the Cas9 molecule.

[0332] In embodiments, the Cas9 molecule comprises the nucleotide sequence of SEQ ID NO: 205 comprising one or more mutations. In some embodiments, the Cas9 mutant is a Streptococcus pyogenes (S. pyogenes) Cas9 mutant. 9 variants contain a mutation at position 147 of SEQ ID NO:205, e.g., 14 of SEQ ID NO:205 containing a tyrosine at position 7 (i.e., SEQ ID NO: 205 with a D147Y mutation, e.g., In an embodiment, the Cas9 variant comprises a nucleotide sequence at position 411 of SEQ ID NO: 205. Mutations include, for example, a threonine at position 411 of SEQ ID NO: 205 (i.e., P4 In an embodiment, the nucleic acid sequence of the present invention comprises, for example, consists of, SEQ ID NO: 205 having a 11T mutation. The Cas9 mutant has a mutation at position 147 and a mutation at position 411 of SEQ ID NO: 205. For example, a tyrosine at position 147 of SEQ ID NO: 205 and a containing a threonine at position 411 (i.e., the D147Y mutation and the P411T mutation) Without being bound by theory, the present invention may be embodied in a variety of ways, including, for example, consisting of, SEQ ID NO: 205. Although such mutations do not improve the targeting efficiency of Cas9 molecules, for example in yeast. In embodiments, the Cas9 molecule comprises a sequence containing one or more mutations. Streptococcus pyogenes (S. pyogenes) Cas9 mutant no. 205. In the Cas9 variant, the Cas9 variant comprises a mutation at position 1135 of SEQ ID NO: 205, e.g., Contains glutamic acid at position 1135 of sequence number 205 (i.e., has the D1135E mutation) Without being bound by theory, However, such mutations are present in the NGG PAM sequence compared to the NAG PAM sequence of the Cas9 molecule. This is thought to improve selectivity for

[0333] In embodiments, the Cas9 molecule contains uncharged or non-polar amino acids at specific positions, e.g. Streptococcus pyogenes (Sp) of SEQ ID NO: 205 containing one or more mutations introducing alanine In embodiments, the Cas9 molecule is a Cas9 mutant of SEQ ID NO: 205 mutation at position 497, mutation at position 661, mutation at position 695 Mutations and / or mutations at position 926, e.g., positions 497 and 661 of SEQ ID NO: 205 205, including mutations at positions 695 and / or 926 to alanine; In embodiments, the Cas9 mutant is a Streptococcus aeruginosa (S. pyogenes) Cas9 mutant. The s9 molecule has amino acids at positions 497, 661, 695, and 696 of SEQ ID NO:205 compared to SEQ ID NO:205. and mutations at only position 926, e.g., where each mutation is an uncharged amino acid, For example, mutation to alanine. Without being bound by theory, These mutations are thought to reduce cleavage of off-target sites by the Cas9 molecule.

[0334] The mutations described herein for the Cas9 molecule can be combined and The Cas9 molecule can be used in combination with any of the fusions or other modifications described herein. It will be understood that the assays described may be tested.

[0335] Various types of Cas molecules may be used in the practice of the invention disclosed herein. In some embodiments, Cas molecules of a type II Cas system are used. In some embodiments, Cas molecules of other Cas systems are used, such as type I or Type III Cas molecules can be used. Exemplary Cas Molecules (and Cas Systems) Haft et al., PLoS COMPUTATIONAL BIOLOGY 2005,1(6):e60 and Makarova et al, NATURE REV IEW MICROBIOLOGY 201 1,9:467-477 (both references , the contents of which are incorporated herein by reference in their entirety.

[0336] In one embodiment, the Cas9 molecule comprises one or more of the following activities: a nickase activity double-strand cleavage activity (e.g., endonuclease and / or exonuclease activity) helicase activity; or the ability to localize to the target nucleic acid together with the gRNA molecule.

[0337] Modified Cas9 molecule Naturally occurring Cas9 molecules have nickase activity, nuclease activity (e.g., endonuclease activity), and nuclease and / or exonuclease activity; helicase activity; gRNA molecule and function the ability to functionally associate with (or localize to) a site on a nucleic acid (e.g., In one embodiment, the antibody has several properties, including, for example, PAM recognition and specificity. A Cas9 molecule may contain all or some of these properties. The as9 molecule interacts with the gRNA molecule and cooperates with the gRNA molecule to localize to the nucleic acid site. Other activities, such as PAM specificity, cleavage activity, or helicase activity, are may vary more widely in the as9 molecule.

[0338] Cas9 molecules with desired properties can be synthesized in a number of ways, e.g., by integrating them into parent, e.g., naturally occurring, Cas9 molecules. To provide a modified Cas9 molecule having desired properties by modifying an existing Cas9 molecule. For example, by introducing one or more mutations or differences compared to the parent Cas9 molecule. Such mutations and differences can include substitutions (e.g., conservative substitutions or non-essential substitutions). amino acid substitutions; insertions; or deletions. In one embodiment, the Cas9 molecule , one or more mutations or differences compared to a reference Cas9 molecule, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 or more mutations, 200, 100, or 80 It may contain less than 10 mutations.

[0339] In some embodiments, the one or more mutations enhance Cas9 activity, e.g., as described herein. In some embodiments, one or Multiple mutations can be made to Cas9 activity, such as the Cas9 activity described herein. In certain embodiments, exemplary activities include PAM specificity, cleavage activity, and the like. and helicase activity. The one or more mutations may, for example, The above RuvC-like domains, such as the N-terminal RuvC-like domain; the HNH-like domain; It may be present in a region outside the vC-like domain and the HNH-like domain. In some embodiments, the one or more mutations are in the N-terminal RuvC-like domain. In some embodiments, the one or more mutations are in the HNH-like domain. Thus, mutations are present in both the N-terminal RuvC-like domain and the HNH-like domain.

[0340] The detailed sequence, e.g., substitutions, may affect one or more activities, such as targeting activity, cleavage activity, etc. Whether a mutation can have an effect on the genome can be determined, for example, by determining whether the mutation is conserved. The amount of the oxidative stress can be determined or predicted by the method described in Section III. In some embodiments, a "non-essential" amino acid residue is one that is used in the context of a Cas9 molecule. When used in a manner that does not abolish Cas9 activity (e.g., cleavage activity), or more preferably or a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, without substantially modifying it. , for example, residues that can be modified from the wild-type sequence of an active Cas9 molecule, while " Alterations in "essential" amino acid residues result in substantial loss of activity (eg, cleavage activity).

[0341] Cas9 molecules with altered or no PAM recognition Naturally occurring Cas9 molecules contain specific PAM sequences, e.g., the PAM sequence of Streptococcus pyogenes (Sp yogenes, S. thermophilus, S. mu tans (S. mutans), Staphylococcus aureus (S. aureus) and Neisseria meningitidis (N. meningitidis) can recognize the PAM recognition sequence described above. Cut.

[0342] In one embodiment, the Cas9 molecule has the same PAM specificity as a naturally occurring Cas9 molecule. In other embodiments, the Cas9 molecule has a similarity to a naturally occurring Cas9 molecule. Unrelated PAM specificity or the naturally occurring Ca with which it has closest sequence homology have PAM specificity unrelated to the Cas9 molecule, e.g., the naturally occurring Cas9 molecule For example, by modifying the PAM recognition, e.g., the PAM recognized by the Cas9 molecule. Modifying the AM sequence to reduce off-target sites and / or improve specificity In some embodiments, the Cas9 molecule can be modified to eliminate the PAM recognition requirement. For example, by increasing the length of the PAM recognition sequence and / or increasing Cas9 specificity. to a high level of identity to reduce off-target sites and improve specificity. In one embodiment, the PAM recognition sequence has a length of at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids in length; recognize different PAM sequences; Cas9 molecules with reduced target and / or off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for the directed evolution of Cas9 molecules are described below. For more information, see, for example, Esvelt el al, Nature 2011, 472 (73 44):499-503. Candidate Cas9 molecules can be prepared, for example, as described herein. It can be evaluated by the method described below.

[0343] Non-cleaving and modified-cleaving Cas9 molecules In one embodiment, the Cas9 molecule is different from a naturally occurring Cas9 molecule, e.g., For example, they contain cleavage properties that differ from those of the most closely homologous naturally occurring Cas9 molecule. For example, the Cas9 molecule may be a naturally occurring Cas9 molecule, such as a Cas9 molecule found in Streptococcus pyogenes (S. pyogenes). genes) as follows: for example, a naturally occurring Cas9 molecules (e.g., the Cas9 molecule of Streptococcus pyogenes (S. pyogenes)). Modulates double-strand break cleavage (endonuclease and / or exonuclease activity) e.g., decreasing or increasing its ability to , a portion of the nucleic acid when compared to the Cas9 molecule of Streptococcus pyogenes (S. pyogenes) Modulating the cleavage of a strand, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity) or nucleic acid molecules, e.g., double-stranded or single-stranded nucleic acids, The ability to cleave the molecule may be removed.

[0344] Modified truncated active Cas9 molecule In some embodiments, an active Cas9 molecule comprises one or more of the following activities: an N-terminal R Cleavage activity associated with the uvC-like domain; Cleavage activity associated with the HNH-like domain; HNH domain Cleavage activity associated with the main and cleavage activity associated with the N-terminal RuvC-like domain.

[0345] In one embodiment, the Cas9 molecule is a Cas9 nickase, e.g., a portion of DNA. In one embodiment, the Cas9 nickase cleaves only one strand of the sequence set forth in SEQ ID NO: 205. 0 and / or 840, e.g., SEQ ID NO: 2 Contains the D10A and / or H840A mutations relative to 05.

[0346] Non-cleavable inactive Cas9 molecule In one embodiment, the modified Cas9 molecule converts the nucleic acid molecule (either a double-stranded nucleic acid molecule or a single-stranded nucleic acid molecule) into a or cleaves nucleic acid molecules with significantly less efficiency, e.g., 20% of the cleavage activity of the reference Cas9 molecule as measured, for example, by the assay described in , an inactive Cas9 molecule that cleaves with an efficiency of less than 10, 5, 1, or 0.1%. The s9 molecule is a naturally occurring unmodified Cas9 molecule, e.g., a Cas9 molecule found in Streptococcus pyogenes (S. pyogenes). genes), S. thermophilus, Staphylococcus aureus Cas9 molecules of S. aureus or N. meningitidis In one embodiment, the reference Cas9 molecule may be a naturally occurring Cas9 molecule, such as a is the naturally occurring Cas9 molecule with the closest sequence identity or homology. In embodiments, the inactive Cas9 molecule is associated with a substantially N-terminal RuvC-like domain. It lacks the cleavage activity and the cleavage activity associated with the HNH-like domain.

[0347] In one embodiment, the Cas9 molecule is dCas9. 2014), Nat.Biotech.32:569-577.

[0348] Catalytically inactive Cas9 molecules can be fused to transcriptional repressors. The protein complexes with the gRNA and targets the DNA specified by the gRNA's targeting domain. It localizes to the A sequence, but unlike active Cas9, it does not cleave the target DNA. Fusing an effector domain, such as a transcriptional repression domain, to an inactive Cas9 gene can inhibit the transcription of gRNs. This allows the effector to be recruited to any DNA site specified by A. Cas9 fusion proteins can be targeted to the promoter region of genes in a site-specific manner. Binding of a polymerase to the promoter region of a transcription factor (e.g., a transcription activator) ) and / or block or affect the binding of transcription enhancers to the nucleic acid. Alternatively, transcriptional activation can be increased or inhibited. Site-specific targeting of Cas9 fusions with lesser to the promoter region of genes can be used to reduce transcriptional activation.

[0349] A transcriptional repressor or transcriptional repressor domain that can be fused to an inactive Cas9 molecule. The Kruppel-associated box (KRAB or SKD), Mad mSIN3 interaction domain The protein may contain a nucleotide sequence encoding ...

[0350] In another embodiment, the inactive Cas9 molecule is fused to a protein that modifies chromatin. For example, an inactive Cas9 molecule may bind to heterochromatin protein 1 (HP1), Histone lysine methyltransferases (e.g., SUV39H1, SUV39H2, G9A, ESET / SETDB l, Pr-SET7 / 8, SUV4-20H1, RIZ 1), histone lysine demethylases (e.g., LSD1 / BHC110, SpLsdl / Sw,l / Safl 10, Su(var)3-3, JMJD2A / JHDM3A, JM JD2B, JMJD2C / GASC1, JMJD2D, Rphl, JARID 1A / R BP2, JARIDIB / PLU-I, JAR1D 1C / SMCX, JARID1D / SMCY, Lid, Jhn2, Jmj2), histone lysine deacetylases (e.g., H DAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hosl, Cir6, HD AC4, HDAC5, HDAC7, HDAC9, HDal, Cir3, SIRT1, SI RT2, Sir2, Hstl, Hst2, Hst3, Hst4, HDAC11) and DN It can be fused to A methylases (DNMT1, DNMT2a / DMNT3b, MET1). Altering the chromatin state using an active Cas9-chromatin modifying molecule fusion protein; The expression of the target gene can be reduced.

[0351] The heterologous sequence (e.g., a transcriptional repressor domain) is attached to the N-terminus of the inactive Cas9 protein. In an alternative embodiment, a heterologous sequence (e.g., a transcriptional repressor) may be fused to the C-terminus. The inactive Cas9 protein domains are located in the internal parts (i.e., other than the N- or C-terminus) of the inactive Cas9 protein. It can be fused to a part.

[0352] The ability of the Cas9 molecule / gRNA molecule complex to bind and cleave the target nucleic acid is For example, the presence or absence of Cas9 can be determined by the methods described in Section III of this specification. a gene, e.g., either an active or inactive Cas9, alone, or a gRNA molecule Activity in complexes with chromatin has also been demonstrated in gene expression assays and chromatin-based assays, e.g. including chromatin immunoprecipitation (ChiP) and chromatin in vivo assay (CiA). This can be determined by methods well known in the art.

[0353] Other Cas9 fusions In embodiments, the Cas9 molecule, e.g., of S. pyogenes, Cas9 may further comprise one or more amino acid sequences that confer additional activities.

[0354] In some embodiments, the Cas9 molecule contains one or more nuclear localization sequences (NLS), e.g., at least Both may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the Cas9 molecule comprises at least one amino acid sequence at or near the amino terminus. Contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS or carboxyl at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more at or near the terminus or a combination thereof (e.g., one or more NLSs at the amino terminus) (One or more NLSs at the base and one or more at the carboxy terminus). When a single NLS is present in two or more copies, each can be selected independently of the other. It may also be present and / or in combination with one or more other NLSs present in one or more copies. In some embodiments, the NLS may be present in combination with the nearest adjacent NLS. The amino acids along the polypeptide chain are approximately 1, 2, 3, 4, 5, 10, 1 N-terminal when it is within the range of 5, 20, 25, 30, 40, 50 amino acids or more NLSs are typically considered to be located near the C-terminus or the NLS. Other types of NLSs consist of one or more short sequences of positively charged lysines or arginines. A non-limiting example of an NLS is the amino acid sequence PKKKRKV (SEQ ID NO: 206) NLS of SV40 virus large T antigen; NL of nucleoplasmin S (e.g., a nucleotide having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 207) Oplasmin bipartite NLS; amino acid sequence PAAKRVKLD (SEQ ID NO: 208 ) or RQRRNELKRSP (SEQ ID NO: 209); QSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(sequence hRNPA1 M9 NLS with the IBB domain of importin-α (number 210) Sequence of RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQIL KRRNV (SEQ ID NO: 211); the sequence of the fibroid T protein VSRKRPRP (SEQ ID NO: 2 12) and PPKKARED (SEQ ID NO: 213); the sequence of human p53 PQPKKKPL ( SEQ ID NO: 214); the sequence SALIKKKKKMAP of mouse c-ab1 IV (SEQ ID NO: 215); influenza virus NS1 sequence DRLRR (SEQ ID NO: 216) and PK QKKRK (SEQ ID NO: 217); sequence of hepatitis virus delta antigen RKLKKKIKKL (sequence Sequence number 218); sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO: 219) ;Sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKK KSKK (SEQ ID NO: 220); and steroid hormone receptor (human) glucocorticoid The sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 221) is included in the Other suitable NLS sequences are known in the art. (e.g., Sorokin, Biochemistry (Moscow) (2007) 72:13,1439-1457;Lange J Biol Chem.(2007) 282:8,5101-5).

[0355] In one embodiment, the Cas9 molecule is a nucleotide sequence encoding a Streptococcus pyogenes (S. pyogenes) polypeptide. ) The Cas9 molecule contains, for example, the SV40 NLS sequence located on the N-terminal side of the Cas9 molecule. In one embodiment, the Cas9 molecule is a nucleotide sequence encoding a Streptococcus pyogenes (S. pyogenes) vector. es) The Cas9 molecule contains the SV40 NLS sequence and CLS sequence located on the N-terminal side of the Cas9 molecule. In one embodiment, the as9 molecule comprises an SV40 NLS sequence located at the C-terminus thereof. Cas9 molecules, such as the S. pyogenes Cas9 molecule, The SV40 NLS sequence is located on the N-terminal side of the Cas9 molecule, and the C-terminal side of the Cas9 molecule. In any of the foregoing embodiments, this molecule comprises the NLS sequence of nucleoplasmin. The molecule may have a tag, for example a His tag, such as a His(6) tag ( The polypeptide may further comprise a His(8) tag (SEQ ID NO: 247) or a His(8) tag (SEQ ID NO: 248).

[0356] In some embodiments, the Cas9 molecule comprises a polypeptide that allows the Cas9 molecule to be specifically recognized. In one embodiment, the tag may include one or more amino acid sequences that Histidine tags, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more In an embodiment, the histidine tag is a histidine tag containing a histidine amino acid. is a His6 tag (6 histidines) (SEQ ID NO: 247). The histidine tag is a His8 tag (8 histidines) (SEQ ID NO: 248). In some embodiments, the histidine tag is attached to one or more other portions of the Cas9 molecule by a linker. In an embodiment, the linker is GGS. An example of such a fusion is C The as9 molecule is iProt106520.

[0357] In some embodiments, the Cas9 molecule comprises one or more enzymes recognized by a protease. In an embodiment, the cleavage site may include an amino acid sequence (e.g., a protease cleavage site). The cleavage site is a tobacco etch virus (TEV) cleavage site, e.g., the sequence ENLYFQG (SEQ ID NO: 230). In some embodiments, the protease cleavage site is The truncation site may be a tag, such as a His tag, for example a His6 (SEQ ID NO: 247) or His8 tag. The Cas9 fragment (SEQ ID NO: 248) is positioned between the fragment and the remainder of the Cas9 molecule. Although not bundled, such introduction allows the tag to be used, for example, for purification of the Cas9 molecule. , which can then be subsequently cleaved so that the tag does not interfere with the function of the Cas9 molecule. It is thought that this is the case.

[0358] In embodiments, a Cas9 molecule (e.g., a Cas9 molecule as described herein) The molecule) contains an N-terminal NLS and a C-terminal NLS (e.g., from the N-terminus to the C-terminus, NLS-C as9-NLS), e.g., where each NLS is an SV40 NLS (PKKKRK V (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (e.g., The Cas9 molecule (as described) contains an N-terminal NLS, a C-terminal NLS, and a C-terminal His6 tag (SEQ ID NO: 247) (e.g., from the N-terminus to the C-terminus, NLS-Cas9-NL S-His tag), e.g., where each NLS is an SV40 NLS (PKKKRK V (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (e.g., The Cas9 molecule as described is tagged with an N-terminal His tag (e.g., His6 tag (SEQ ID NO: No. 247)), and contains an N-terminal NLS and a C-terminal NLS (e.g., from the N-terminus to the C-terminus, His-tag-NLS-Cas9-NLS), e.g., where each NLS is SV40 In an embodiment, the Cas9 gene is a Cas9 NLS (PKKKRKV (SEQ ID NO: 206)). The molecule (e.g., a Cas9 molecule as described herein) has an N-terminal NLS and a C-terminal H including an is tag (e.g., a His6 tag (SEQ ID NO: 247)) (e.g., from the N-terminus to the C-terminus at the end, containing a His tag-Cas9-NLS), e.g., where NLS is SV40 NLS. LS(PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule ( For example, a Cas9 molecule as described herein may contain an N-terminal NLS and a C-terminal His A tag (e.g., a His6 tag (SEQ ID NO: 247)) is included (e.g., from the N-terminus to the C-terminus). , including an NLS-Cas9-His tag), e.g., where NLS is SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (e.g., For example, a Cas9 molecule as described herein) may be tagged with an N-terminal His tag (e.g., His 8 tag (SEQ ID NO: 248)), an N-terminal cleavage domain (e.g., tobacco etch virus (T EV) cleavage domain (e.g., comprising the sequence ENLYFQG (SEQ ID NO: 230)), N-terminus a terminal NLS (e.g., SV40 NLS; SEQ ID NO: 206), and a C-terminal NLS (e.g., SV40 NLS; SEQ ID NO: 206) (e.g., from the N-terminus to the C-terminus, a His tag In any of the foregoing embodiments, Ca s9 has the sequence of SEQ ID NO: 205. Alternatively, in any of the foregoing embodiments, C as9 is the sequence of the Cas9 mutant of SEQ ID NO: 205, e.g., as described herein. In any of the foregoing embodiments, the Cas9 molecule comprises a His tag and a sequence of The exemplary Ca moieties described above may include a linker between them, such as a GGS linker. The amino acid sequence of the s9 molecule is provided below. The "iProt" identifiers correspond to those in Figure 60. do. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0359] Nucleic acid encoding the Cas9 molecule Nucleic acids encoding Cas9 molecules, e.g., active or inactive Cas9 molecules, are included herein. Provided in the specification.

[0360] Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al., SCIENCE 2 013,399(6121):819-823;Wang et al.CELL 20 13,153(4):910-918;Mali et al., SCIENCE 20 13,399(6121):823-826;Jinek et al. 2012,337(6096):816-821.

[0361] In some embodiments, the nucleic acid encoding the Cas9 molecule can be a synthetic nucleic acid sequence. For example, synthetic nucleic acid molecules can be chemically modified, e.g., as described in Section XIII. In some embodiments, the Cas9 mRNA exhibits one or more of the following properties, including totality: It has the following characteristics: it is capped, polyadenylated, 5-methylsuccinimide It is substituted with uridine and / or pseudouridine.

[0362] Additionally or alternatively, the synthetic nucleic acid sequence may be codon optimized, e.g., to include at least one codon A rare or low frequency codon is replaced by a high frequency codon, e.g. The synthetic nucleic acids may be, for example, optimized for expression in a mammalian expression system, e.g., as described herein. This can lead to the synthesis of optimized messenger mRNA.

[0363] Below is an exemplary sequence encoding the Cas9 molecule of S. pyogenes: Codon-optimized nucleic acid sequences are provided. [ka] [ka] [ka] [ka]

[0364] Below is an exemplary codon-optimized nucleic acid sequence encoding a Cas9 molecule comprising SEQ ID NO: 244: to provide. [ka] [ka] [ka]

[0365] When the above Cas9 sequence is fused to a peptide or polypeptide at the C-terminus (e.g., (Inactive Cas9 fused to a transcriptional repressor at the C-terminus), the stop codon is removed. It is understood that

[0366] Nucleic acids, vectors for the production of Cas9 molecules, e.g., the Cas9 molecules described herein Recombinant production of polypeptide molecules is well within the skill of the art. This can be done using known techniques, for example, Cas9 molecules as described herein. Described herein are molecules and methods for the recombinant production of any polypeptide molecule. As used in the context of this specification, "recombinant" molecules and production refers to the production of molecules encoding the molecule of interest. isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the nucleic acid The isolated polypeptide, the hybridomas prepared therefrom, and the hybridomas expressing this molecule From transformed host cells, e.g., from transfectomas molecules isolated from recombinant, combinatorial libraries, and molecules Splicing all or part of the gene encoding (or part of) another DNA sequence or molecules prepared, expressed, produced or isolated by any other means, including by cloning. Any polypeptide prepared, expressed, produced or isolated by recombinant means (e.g., Recombinant production involves the use of host cells, e.g., Cas9 molecules as described herein. For example, a molecule described herein, such as a Cas9 molecule, for example a Cas described herein. The vector may be derived from a host cell containing nucleic acid encoding the vector.

[0367] For example, a molecule as described herein (e.g., a Cas9 molecule and / or a gRN Nucleic acid molecules encoding the A molecule are provided herein. 244, or any one of SEQ ID NOs: 233 to 244. or a fragment thereof, or a fragment thereof having at least one of SEQ ID NOs: 233 to 244. 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98%, or less Specifically, nucleic acid molecules containing sequences encoding polypeptides with at least 99% sequence identity are will be provided to.

[0368] A vector, e.g., as described herein, comprising any of the above nucleic acid molecules is In embodiments, the nucleic acid molecule comprises a promoter, e.g. The vector is operably linked to a promoter that is operable in the host cell into which it is introduced.

[0369] Host cells containing one or more of the nucleic acid molecules and / or vectors described herein are In embodiments, the host cell is a prokaryotic host cell. In some embodiments, the host cell is a eukaryotic host cell. In an embodiment, the host cell is a mammalian cell. Such host cells are suitable for expressing the recombinant molecules described herein, e.g., human cells. For example, they can be used to produce Cas9 or gRNA molecules as described herein. do.

[0370] VI. Functional analysis of candidate molecules The candidate Cas9 molecules, candidate gRNA molecules, and candidate Cas9 molecule / gRNA molecule complexes are may be assessed by methods known in the art or as described herein. For example, an exemplary method for assessing the endonuclease activity of a Cas9 molecule is Jinek el al., SCIENCE 2012;337(6096):81 6-821.

[0371] VII. Template nucleic acid (for nucleic acid transfer) The term "template nucleic acid" or "donor template" as used herein refers to a nucleic acid fragment of the present invention. Nucleic acid inserted at or near the target sequence modified by the SPR system, e.g., cleaved In one embodiment, a nucleic acid sequence at or near a target sequence is typically The modified nucleic acid fragments are modified to have part or all of the sequence of the template nucleic acid at or near one or more cleavage sites. In certain embodiments, the template nucleic acid is single-stranded. The template nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, e.g., double-stranded DNA. A. In an alternative embodiment, the template nucleic acid is single-stranded DNA.

[0372] In embodiments, the template nucleic acid encodes a globin protein, such as beta-globin. In some embodiments, the nucleic acid encodes a sequence encoding the β-globin gene. The beta globin to be delivered contains one or more mutations, e.g., anti-sickling mutations. In some embodiments, the beta globin encoded by the nucleic acid comprises the mutation T87Q. In some embodiments, the beta globin encoded by the nucleic acid contains the mutation G16D. In an embodiment, the beta globin encoded by the nucleic acid comprises the mutation E22A. In an embodiment, the beta globin gene contains the mutations G16D, E22A and T87Q. In embodiments, the template nucleic acid contains one or more regulatory elements, e.g., a promoter. (e.g., human β-globin promoter), 3' enhancer, and / or globin gene At least a portion of a locus control region (e.g., one or more DNase I hypersensitive sites (e.g., For example, HS2, HS3 and / or HS4 of the human globin locus.

[0373] In other embodiments, the template nucleic acid comprises a sequence encoding gamma globin, e.g., gamma globin. In an embodiment, the template nucleic acid contains two or more gamma globin genes. and / or a sequence encoding a copy of the gamma globin gene, e.g., two or more, e.g., two gamma globin gene sequences. In embodiments, the template nucleic acid contains one or more regulatory elements, e.g., a promoter. and / or enhancers.

[0374] In some embodiments, the template nucleic acid is selected by participating in a homology-directed repair event. Modifying the structure of the target location. In some embodiments, the template nucleic acid is In some embodiments, the template nucleic acid is a modified or non-naturally occurring nucleic acid. This results in the incorporation of a base.

[0375] Mutations in the genes or pathways described herein may be used in the methods discussed herein. In one embodiment, the genes or pathways described herein can be modified using one of the following: Mutations in the gene are corrected by homology-directed repair (HDR) using a template nucleic acid. In some embodiments, mutations in the genes or pathways described herein are mediated by a template In one embodiment, the gene is corrected by homologous recombination (HR) using a nucleic acid. Mutations in the genes or pathways described herein can be made by non-homologous end joining (NHL) using a template nucleic acid. In another embodiment, the nucleic acid encoding the molecule of interest is corrected by NHEJ repair. The acid is inserted at or near the site modified by the CRISPR system of the present invention. In embodiments, the template nucleic acid is a nucleic acid sequence that is a target molecule, e.g., as described herein. a regulatory element, e.g., one or more promoters, operably linked to the nucleic acid sequence encoding the gene; Includes a motor and / or enhancer.

[0376] HDR or HR repair and template nucleic acid As described herein, nuclease-directed homology-directed repair (HDR) or homologous recombination repair (HDR) is used. Recombination (HR) is used to modify target sequences and correct genomic mutations (e.g., repair Without wishing to be bound by theory, The alteration of the sequence may occur by repair based on a donor template or template nucleic acid. For example, a donor template or template nucleic acid may result in modification of the target sequence. It is contemplated that a double-stranded template can be used as a template for homologous recombination. - Alternative homology-directed repair methods (e.g., single-stranded amino acid sequence repair) between the target sequence and the donor template It is contemplated that the donor template may be used as a template for modification of the target sequence by ligation. The modification of the target sequence achieved by Cas9 may depend on cleavage by the Cas9 molecule. The cleavage by can include a double-strand break, one single-strand break, or two single-strand breaks.

[0377] In one embodiment, the mutation is either a single double-strand break or two single-strand breaks. In one embodiment, the mutation can be: (1) a double (2) two single-strand breaks; (3) two double-strand breaks, each resulting in a break on either side of the target sequence. (4) one double-strand break resulting in a double-strand break and two single-strand breaks on each side of the target sequence and two single-strand breaks, (5) four single-strand breaks resulting in a pair of single-strand breaks on each side of the target sequence. (6) a CRISPR / Cas9 system and template that creates a single-strand break; This can be corrected by providing

[0378] Double-strand break-mediated repair In one embodiment, the cleavage activity associated with the HNH-like domain and the RuvC-like domain, For example, a Cas9 molecule having cleavage activity associated with an N-terminal RuvC-like domain, e.g., A double-stranded break is achieved by live Cas9. Such an embodiment requires only a single gRNA. is necessary.

[0379] Single-strand break-mediated repair In other embodiments, nickase activity, e.g., cleavage activity associated with an HNH-like domain. or by Cas9 molecules with cleavage activity associated with the N-terminal RuvC-like domain. Single-strand breaks, or nicks, are achieved. Such embodiments include the placement of each single-strand break. In one embodiment, two gRNAs are required, one for each gene. The Cas9 molecule cleaves the strand to which the gRNA hybridizes, It does not cleave the strand complementary to the strand that it cleaves. The Cas9 molecule does not cleave the strand to which the gRNA is hybridized, but rather cleaves the strand to which the gRNA is hybridized. The strand complementary to the hybridizing strand is cleaved.

[0380] In one embodiment, the nickase has HNH activity, e.g., RuvC activity is inactive. The Cas9 molecule being activated, e.g., a mutation in D10, e.g., a D10A mutation, D10A inactivates RuvC. s9 nickase has HNH activity (only) and selectively hybridizes to the strand to which the gRNA hybridizes (e.g. In other embodiments, the complementary strand (which does not have an NGG PAM therein) will be cleaved. and using a Cas9 molecule with an H840, e.g., H840A, mutation as a nickase. H840A inactivates HNH. Therefore, Cas9 nick The enzyme has RuvC activity (only) and a non-complementary strand (e.g., NGG PAM and its The strand whose sequence is identical to the gRNA is cut.

[0381] In an embodiment where a nickase and two gRNAs are used to place two single-stranded nicks, In this case, one nick is on the + strand and one nick is on the - strand of the target nucleic acid. M faces outward. The gRNA is approximately 0-50, 0-100, or 0-2 In one embodiment, the sequences can be selected to be separated by 0 nucleotides. There is no overlap between the target sequences complementary to the targeting domains of the two gRNAs. In embodiments, the gRNAs are non-overlapping and are 50, 100, or 200 nucleotides long. In one embodiment, two gRNAs are used, e.g., This may increase specificity by reducing off-target binding (Ran et al. ,CELL 2013).

[0382] In certain embodiments, a single nick can be used to induce HDR. A single nick can be used to increase the rate of HDR, HR, or NHEJ at a given cleavage site. It is intended that

[0383] Positioning of double-stranded or single-stranded breaks relative to the target location The double-stranded or single-stranded break in one of the strands is located sufficiently close to the target position for correction to occur. In some embodiments, the distance is 50, 100, 200, 30 0, 350, or 400 nucleotides or less. Although not all breakpoints are involved in exonuclease-mediated removal of the breakpoints during terminal resection, It is believed that the target location must be close enough to the target location so that it is within the area that receives the signal. The target position can be determined by the fact that the core sequence can only be used to modify the sequence within the terminal resection region. If the distance between the position and the breakpoint is too great, the terminal resection will not contain the mutation. may be too slow and therefore may not be corrected.

[0384] gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease In embodiments where the double-strand break is induced for the purpose of inducing HDR- or HR-mediated correction, In this case, the cleavage site is 0 to 200 bp (e.g., 0 to 175, 0 to 150, 0 ~125, 0~100, 0~75, 0~50, 0~25, 25~200, 25~175, 25~150, 25~125, 25~100, 25~75, 25~50, 50~200, 50~175, 50~150, 50~125, 50~100, 50~75, 75~200 , 75-175, 75-150, 75-125, 75-100 bp). In some embodiments, the cleavage site is located 0 to 100 bp (e.g., 0 to 75, 0 to 100 bp) from the target position. 50, 0~25, 25~100, 25~75, 25~50, 50~100, 50~75 are separated by 75-100 bp).

[0385] Two gRNAs (independently, monomolecular (or chimeric) or (is a modular gRNA) induces two single-strand breaks to induce HDR-mediated repair. In some embodiments, the closer nick is 0-200 bp (e.g., 0-1 75, 0~150, 0~125, 0~100, 0~75, 0~50, 0~25, 25~2 00, 25~175, 25~150, 25~125, 25~100, 25~75, 25~ 50, 50-200, 50-175, 50-150, 50-125, 50-100, 50 ~75, 75~200, 75~175, 75~150, 75~125, 75~100bp ), and the two nicks are ideally within 25-55 bp of each other (e.g., 25~50, 25~45, 25~40, 25~35, 25~30, 30~55, 30 ~50, 30~45, 30~40, 30~35, 35~55, 35~50, 35~45, 35-40, 40-55, 40-50, 40-45 bp), and each is within 100 bp (e.g., 90, 80, 70, 60, 50, 40, 30, 20, 1 In one embodiment, the cleavage site is 0 or 5 bp or less from the target position. ~100 bp (e.g., 0–75, 0–50, 0–25, 25–100, 25–75, 2 5-50, 50-100, 50-75, or 75-100 bp apart.

[0386] In one embodiment, two gRNAs, e.g., independently, are used as monomolecules (or chimeras) or monoclonals. The transducing gRNA is configured to place a double-stranded break on either side of the target location. In the form of three gRNAs, e.g., independently, monomolecular (or chimeric) or modular The gRNAs generate double-stranded breaks on either side of the target location (i.e., one gRNA is Cas9 complexed with a nuclease), and two single-strand breaks or paired single-strand breaks (i.e., The two gRNAs are configured to complex with the Cas9 nickase (e.g., For example, a first gRNA is used to target the upstream (i.e., 5') side of the target location, and a second gRNA is used to target the target location. In another embodiment, NA is used to target downstream (i.e., 3') of the target position. , four gRNAs, e.g., independently, monomolecular (or chimeric) or modular gRNAs, Generate two pairs of single-strand breaks on either side of the target location (i.e., two pairs of two gRNs) A is configured to complex with the Cas9 nickase (e.g., the first gRNA A first gRNA is used to target the upstream (i.e., 5') side of the target location, and a second gRNA is used to target the target location targeting the downstream (i.e., 3') side of a double-stranded break or two single-stranded nicks in a pair. The closer of the two blocks is ideally within 0–500 bp of the target position (e.g., From the position 450, 400, 350, 300, 250, 200, 150, 100, 50 or 2 When a nickase is used, the two nicks in the pair are aligned with each other. Within 25-55bp (e.g., 25-50, 25-45, 25-40, 25-35, 25~30, 50~55, 45~55, 40~55, 35~55, 30~55, 30~5 0, 35-50, 40-50, 45-50, 35-45, or 40-45 bp), Within 100 bp of each other (e.g., 90, 80, 70, 60, 50, 40, 30, 20 or 10bp or less).

[0387] In one embodiment, two gRNAs, e.g., independently, are used as monomolecules (or chimeras) or monoclonals. The transducing gRNA is configured to place a double-stranded break on either side of the target location. In the form of three gRNAs, e.g., independently, monomolecular (or chimeric) or modular The gRNA makes double-stranded breaks in two target sequences (i.e., one gRNA is inserted into the Cas9 nucleus). complex with the enzyme) and two single-strand breaks or paired single-strand breaks (i.e., two g The RNA is complexed with the Cas9 nickase (e.g., a first A gRNA is used to target a target sequence upstream (i.e., 5') of the insertion site, and a second gR In another embodiment, the NA is used to target a downstream (i.e., 3') target sequence. , four gRNAs, e.g., independently, monomolecular (or chimeric) or modular gRNAs, Generate two pairs of single-strand breaks on either side of the insertion site (i.e., two pairs of two gRN A is configured to complex with the Cas9 nickase (e.g., the first gRNA to target the upstream (i.e., 5') target sequence described herein, and a second gR NAs are used to target downstream (i.e., 3') target sequences described herein). The closer of the two single-stranded nicks in the double-stranded break or pair is ideally at the target site. Within 0 to 500 bp from the target position (e.g., 450, 400, 350, 300, 2 (50, 200, 150, 100, 50 or 25 bp or less). Nickase is used. If a pair is used, the two nicks in the pair should be within 25-55 bp of each other (e.g., 25-50, 25~45, 25~40, 25~35, 25~30, 50~55, 45~55, 40~5 5, 35-55, 30-55, 30-50, 35-50, 40-50, 45-50, 35 ~45, or 40-45 bp) and are within 100 bp of each other (e.g., 90, 80, 7 0, 60, 50, 40, 30, 20, or 10 bp apart).

[0388] Homology arm length The homology arms may be, for example, resectioned single-stranded overhangs that are complementary to the homologous sequences in the donor template. To be able to find the complementary region, at least the region where terminal resection can occur The total length must be the same as the plasmid size or viral package. In some embodiments, the homology archiving may be limited by parameters such as binding limits. The template does not extend into repetitive elements, e.g., ALU repeats, LINE repeats. may have two homology arms of the same or different lengths.

[0389] Exemplary homology arm lengths are at least 25, 50, 100, 250, 500, 750, or 1000 nucleotides.

[0390] A target location, as used herein, refers to a site that is modified by a Cas9 molecule-dependent process. For example, a target location refers to a site on a target nucleic acid (e.g., a chromosome) where a modified Cas9 molecule and modification, e.g., correction, of the target site guided by the template nucleic acid. In one embodiment, the target position is a target nucleotide to which one or more nucleotides are added. The target position can be between two nucleotides on the base, for example, between adjacent nucleotides. The template nucleic acid may contain one or more nucleotides that are modified, e.g., altered, by the template nucleic acid. In embodiments, the target position is within the target sequence (e.g., the sequence to which the gRNA binds). In one embodiment, the target position is the position of the target sequence (e.g., the sequence to which the gRNA binds). It is upstream or downstream.

[0391] Typically, the template sequence undergoes cleavage-mediated or catalytic recombination with the target sequence. In embodiments, the template nucleic acid comprises a target sequence that is cleaved by a Cas9-mediated cleavage event. In some embodiments, the template nucleic acid comprises a sequence corresponding to a site on the array. a first site on the target sequence that is cleaved in a Cas9-mediated event and a second Cas9-mediated event and a second site on the target sequence cleaved at that site.

[0392] In some embodiments, the template nucleic acid comprises a sequence that results in the modification of the coding sequence of the translation sequence: For example, substitution of one amino acid for another in a protein product, e.g., a mutant allele. converting a wild-type allele to a mutant allele, converting a wild-type allele to a mutant allele, and / or Substitutions that introduce stop codons, insertions of amino acid residues, deletions of amino acid residues, or nonsense mutations It may contain sequences that give rise to natural mutations.

[0393] In other embodiments, the template nucleic acid may include modifications of non-coding sequences, such as exons or 5' or sequences that result in modifications of the 3' untranslated or untranscribed region. Modification of regulatory elements, such as promoters, enhancers, and cis-acting or trans-acting genes. Modifications of the gene expression control elements are included.

[0394] The template nucleic acid can include a sequence that, when incorporated, results in: Decreased activity of positive regulatory elements; Increased activity of positive regulatory elements; reduced activity of negative regulatory elements; Increased activity of negative regulatory elements; Decreased gene expression; Increased gene expression; Increased resistance to injury or disease; Increased resistance to viral invasion; Correction of mutations or modification of undesired amino acid residues; Imparting, increasing, eliminating, or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme , or an increase in the ability of the gene product to interact with another molecule.

[0395] The template nucleic acid can include a sequence that gives rise to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence sequence changes exceeding

[0396] In some embodiments, the template nucleic acid may be 20±10, 30±10, 40±10, 50±1 0, 60±10, 70±10, 80±10, 90±10, 100±10, 110±10, 120±10, 130±10, 140±10, 150±10, 160±10, 170±1 0, 180±10, 190±10, 200±10, 210±10, 220±10, 200 ~300, 300~400, 400~500, 500~600, 600~700, 700 ~800, 800~900, 900~1000, 1000~2000, 2000~300 0 or more than 3000 nucleotides in length.

[0397] The template nucleic acid comprises the following components: [5' homology arm]-[insertion sequence]-[3' homology arm].

[0398] The homology arms allow for recombination into the chromosome, thereby removing the unwanted element, For example, the mutation or signature can be replaced with a replacement sequence. In this case, the homology arms flank the most distal cleavage site.

[0399] In one embodiment, the 3' end of the 5' homology arm is located adjacent to the 5' end of the replacement sequence. In one embodiment, the 5' homology arm is located 5' from the 5' end of the replacement sequence. At least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, It may extend for 1000, 1500, or 2000 nucleotides.

[0400] In one embodiment, the 5' end of the 3' homology arm is located adjacent to the 3' end of the replacement sequence. In one embodiment, the 3' homology arm is located 3' from the 3' end of the replacement sequence. At least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, It may extend for 1000, 1500, or 2000 nucleotides.

[0401] As used herein, shortening one or both homology arms to form a particular sequence repeat is referred to as a "sequence repeat." elements, e.g., Alu repeats, LINE elements, may be avoided. For example, shortening the 5' homology arm to avoid sequence repeat elements is contemplated. In other embodiments, the 3' homology arm may be shortened to avoid sequence repeat elements. In some embodiments, both the 5' and 3' homology arms may be shortened to provide a specific sequence linkage. The inclusion of peat elements can be avoided.

[0402] As used herein, the template nucleic acid for mutation correction is a single-stranded oligonucleotide (ssODN). It is contemplated that ssODNs can be designed to be used as 5 The 1' and 3' homology arms can be up to about 200 base pairs (bp) in length, e.g., at least 25, 50 , 75, 100, 125, 150, 175, or 200 bp in length. For sODNs, longer homology sequences are required as oligonucleotide synthesis continues to improve. Arms are also contemplated.

[0403] NHEJ method for gene targeting As described herein, nuclease-induced non-homologous end joining (NHEJ) is used. Nuclease-induced NHEJ can be used to target gene-specific knockout. It can also be used to remove (eg, delete) sequences of a gene of interest.

[0404] Without wishing to be bound by theory, in certain embodiments, The genome modifications associated with the methods described in are nuclease-induced NHEJ and NHEJ repair. NHEJ is thought to rely on the error-prone nature of the reverse pathway. The strand break is repaired by splicing the ends together. , two compatible ends are ligated together as they are formed by the double-strand break The original sequence is restored only if the DNA ends of the double-strand break are frequently resistant to enzymatic processing. nucleotides are added to one or both strands before the ends are rejoined to form an image. This results in the addition or removal of insertions and / or deletions in the DNA sequence at the NHEJ repair site. Deletion (indel) mutations will be present. Two-thirds of these mutations are lead This can alter the reading frame and thus result in a non-functional protein. Mutations that maintain the coding frame but insert or delete large amounts of sequence can alter the protein structure. Mutations in important functional domains can disrupt the function of a protein. This is locus-dependent, as mutations in the nucleobase region are likely to be less tolerated than in the nucleobase region. be.

[0405] Indel mutations created by NHEJ are inherently unpredictable. However, at a given breakpoint, certain indel sequences are preferred and overrepresented in the ...

Claims

1. A gRNA molecule comprising a tracr and a crRNA, wherein the crRNA is a) Chr11: 5,250,094-5,250,237, -strand, Gel in hg38 is complementary to a target sequence within the genome nucleic acid sequence; b) Chr11: 5,255,022 to 5,255,164, -strand, Gel in hg38 is complementary to a target sequence within the genome nucleic acid sequence; c) complementary to a target sequence in a non-deleted HFPH region (e.g., a human non-deleted HFPH region); ； d) Chr11:5,249,833 to Chr11:5,250,237, -chain, hg3 8 is complementary to a target sequence within the genomic nucleic acid sequence; e) Chr11:5,254,738 to Chr11:5,255,164, -chain, hg3 8 is complementary to a target sequence within the genomic nucleic acid sequence; f) Chr11: 5,249,833-5,249,927, -chain, Gel in hg38 is complementary to a target sequence within the genome nucleic acid sequence; g) Chr11: 5,254,738 to 5,254,851, -chain, genomic DNA in hg38 is complementary to a target sequence within the genome nucleic acid sequence; h) Chr11: 5,250,139 to 5,250,237, -chain, Gel in hg38 is complementary to a target sequence within the nucleic acid sequence; or i) Combinations of these A gRNA molecule comprising a targeting domain which is

2. The targeting domain comprises any one of SEQ ID NO: 1 to SEQ ID NO: 72 or a fragment thereof.

2. The gRNA molecule of claim 1, for example consisting of:

3. The targeting domain may be any of SEQ ID NO:67, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: No. 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: No. 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63 or a fragment thereof 3. The gRNA molecule of claim 2, comprising, for example consisting of, any one of:

4. The targeting domain comprises: a) SEQ ID NO: 67, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 4 8. SEQ ID NO: 51 or a fragment thereof; or b) SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 54 or fragments thereof 3. The gRNA molecule of claim 2, comprising, for example consisting of, any one of:

5. The targeting domain may be any one of the targeting domain sequences described above. Any of claims 2 to 4, comprising, for example consisting of, 8, 19 or 20 consecutive nucleic acids.

1. A gRNA molecule according to claim 1.

6. the 17, 18, 19, or 20 sequences of any one of the targeting domain sequences described above; The contiguous nucleic acids are located at the 3' end of the targeting domain sequence described above. The gRNA molecule of claim 5, which is 8, 19, or 20 consecutive nucleic acids.

7. the 17, 18, 19, or 20 sequences of any one of the targeting domain sequences described above; The contiguous nucleic acids are located at the 5' end of the targeting domain sequence described above. The gRNA molecule of claim 5, which is 8, 19, or 20 consecutive nucleic acids.

8. the 17, 18, 19, or 20 sequences of any one of the targeting domain sequences described above; The contiguous nucleic acids include either the 5' or 3' nucleic acids of the targeting domain sequence described above. The gRNA molecule of claim 5 .

9. The targeting domain of any one of claims 2 to 8, wherein the targeting domain consists of the targeting domain sequence described above.

1. The gRNA molecule of any one of claims 1 to 9.

10. 10. The method of claim 1, wherein the targeting domain comprises, for example consists of, SEQ ID NO:

67. The gRNA molecule of any one of claims 1 to 4.

11. The gRNA molecule according to any one of claims 1 to 10, which is a dual guide RNA molecule. child.

12. The gRNA molecule according to any one of claims 1 to 10, which is a single guide RNA molecule. child.

13. (a) SEQ ID NO: 195; (b) SEQ ID NO: 231; or (c) adding 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides to the 3' end; Either (a) or (b) above and wherein the sequence of any of (a) to (c) is optionally located immediately 3′ of said targeting domain. The gRNA molecule of claim 12, wherein the gRNA molecule is positioned on the '-side.

14. array: (a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ ID NO: 176 2. The gRNA molecule of claim 1, comprising, for example consisting of:

15. (a) a crRNA comprising, for example consisting of, SEQ ID NO: 177 and a crRNA comprising, for example, SEQ ID NO: 224 hmm, for example, tracr consisting of it; (b) a crRNA comprising, for example consisting of, SEQ ID NO: 177 and comprising SEQ ID NO: 73; For example, tracr consisting of: (c) a crRNA comprising, for example consisting of, SEQ ID NO: 178 and comprising SEQ ID NO: 224 , e.g., a tracr consisting thereof; or (d) a crRNA comprising, for example consisting of, SEQ ID NO: 178 and comprising SEQ ID NO: 73; For example, tracr consisting of 2. The gRNA molecule of claim 1, comprising, for example consisting of:

16. a) a CRISPR system (e.g., as described herein) comprising the gRNA molecule; When the gRNA molecule (RNP) is introduced into a cell, the indels are inserted into the targeting domain of the gRNA molecule. formed at or near a target sequence complementary to the target sequence; and / or b) a CRISPR system (e.g., as described herein) comprising the gRNA molecule; When introduced into a cell, the RNP (i.e., the RNP) contains, for example, substantially all of the sequence. The deletion is complementary to the gRNA targeting domain (e.g., For example, at least 90% complementary to the gRNA targeting domain, e.g., at least 90% complementary to the gRNA targeting domain. a sequence (fully complementary to the HBG2 promoter region) and the gRNA target sequence in the HBG2 promoter region. complementary to the gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain) , e.g., fully complementary to the gRNA targeting domain), Item 16. The gRNA molecule according to any one of items 1 to 15.

17. The indel is located between 5,250,092 and 5,249,833, and between the -strand (hg38). and optionally, the indel does not include a nucleotide located at the end of the non-deleted HPFH or transcriptional The gRNA molecule of claim 16, which does not include nucleotides of a factor binding site.

18. A CRISPR system (e.g., as described herein) comprising the gRNA molecule When a RNP (RNP) is introduced into a population of cells, indels are generated in at least one of the cells of the population. about 15%, for example at least about 17%, for example at least about 20%, for example at least about 30%, for example at least about 40%, for example at least about 50%, for example at least about 5 5%, for example at least about 60%, for example at least about 70%, for example at least about 75 % at or near the target sequence complementary to the targeting domain of the gRNA molecule The gRNA molecule of any one of claims 1 to 17, wherein the gRNA molecule is formed as follows:

19. A CRISPR system (e.g., as described herein) comprising the gRNA molecule When introduced into a cell (e.g., a population of cells), (a) expression of fetal hemoglobin is in the cells or their progeny, e.g., their erythroid progeny, e.g., For example, the expression of fetal hemoglobin is increased in the red blood cell progeny thereof, and optionally, a population of cells into which the gRNA molecule has not been introduced or their progeny, e.g., their erythroid progeny to the level of expression of fetal hemoglobin in the population of their red blood cell progeny, e.g. , at least about 15%, for example at least about 17%, for example at least about 20%, for example At least about 25%, for example at least about 30%, for example at least about 35%, for example at least about increased by at least about 40%; (b) said cell or population of cells or their progeny, e.g., their erythroid progeny, e.g., their red blood cells; The sphere cell progeny have at least about 6 picograms (e.g., at least about 7 picograms) per cell. gram, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms 10 picograms, or about 8 to about 9 picograms, or about 9 to about 10 picograms) of fetal hemoglobin Produce; (c) off-target indels are not formed in the cell, e.g., in the next generation Offta as detectable by sequencing and / or nucleotide insertion assays The target indel is formed outside the HBG1 and / or HBG2 promoter region. and / or (d) detection, for example, by next-generation sequencing and / or nucleotide insertion assays Possible off-target indels, such as the HBG1 and / or HBG2 promoters off-target indels outside the target region in more than about 5% of the cells in the population of cells; For example, not detected in more than about 1%, for example, more than about 0.1%, for example, more than about 0.01%.

19. The gRNA molecule of any one of claims 1 to 18.

20. The cell may be a mammalian cell, a primate cell or a human cell, for example a human cell (or a population of cells comprising the same), and optionally the cells are a hemoglobinopathy, e.g. obtained from a patient suffering from sickle cell disease or thalassemia, e.g., β-thalassemia; A gRNA molecule according to any one of claims 16 to 19.

21. The cells are HSPCs, optionally CD34+ HSPCs, optionally CD34+ CD9 0+ HSPCs. child.

22. The cells are autologous or allogeneic to the patient receiving the cells.

22. The gRNA molecule of any one of claims 16 to 21.

23. 1) one or more gRNA molecules according to any one of claims 1 to 22 (first gRNA molecule) and a Cas9 molecule; 2) One or more gRNA molecules according to any one of claims 1 to 22 (first gRNA molecule a nucleic acid encoding a Cas9 molecule; 3) One or more gRNA molecules according to any one of claims 1 to 22 (first gRNA molecule a nucleic acid encoding a Cas9 molecule; 4) One or more gRNA molecules according to any one of claims 1 to 22 (first gRNA molecule a nucleic acid encoding a Cas9 molecule; 5) any one of 1) to 4) above and a template nucleic acid; or 6) A nucleic acid comprising a sequence encoding any one of the above 1) to 4) and a template nucleic acid. A composition comprising:

24. A method for producing a nucleic acid sequence comprising the first gRNA molecule according to any one of claims 1 to 22, and further comprising the step of: and optionally, the Cas9 molecule is an active or inactivated Streptococcus pylori. s. pyogenes Cas9, and optionally the Cas9 molecule has the sequence SEQ ID NO:205 or at least 95%, 96%, 97%, 98% or 99% of the sequence A composition comprising a sequence having homology.

25. The Cas9 molecule (a) SEQ ID NO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e) SEQ ID NO: 237; (f) SEQ ID NO: 238; (g) SEQ ID NO: 239; (h) SEQ ID NO: 240; (i) SEQ ID NO: 241; (j) SEQ ID NO: 242; (k) SEQ ID NO: 243; or (l) SEQ ID NO: 244 25. A composition according to claim 23 or 24, comprising, for example consisting of:

26. The first gRNA molecule and the Cas9 molecule are contained in a ribonucleoprotein complex (RNP). The composition of any one of claims 23 to 25, wherein

27. Any of claims 23 to 26, formulated in a medium suitable for electroporation. The composition described in claim 1.

28. Each of the gRNA molecules is in an RNP with a Cas9 molecule as described herein. , each of said RNPs is less than about 10 uM, e.g., less than about 3 uM, e.g., less than about 1 uM, e.g., For example, a concentration of less than about 0.5 uM, for example less than about 0.3 uM, for example less than about 0.1 uM. Optionally, the concentration of the RNP is about 2 uM or about 1 uM; and optionally Optionally, the composition further comprises a population of cells, e.g., HSPCs. The composition according to any one of claims 1 to 4.

29. A nucleic acid sequence encoding one or more gRNA molecules according to any one of claims 1 to 22. 。

30. 30. A vector comprising the nucleic acid of claim 29, optionally a lentiviral vector. -, adenovirus vector, adeno-associated virus (AAV) vector, herpes simplex Virus (HSV) vectors, plasmids, minicircles, nanoplasmids and RNA vectors A vector selected from the group consisting of:

31. A cell (e.g., a population of cells) is modified at or near a target sequence within the cell (e.g., a method for modifying the structure (e.g., sequence) of a nucleic acid, the method comprising: group), 1) one or more gRNA molecules and a Cas9 molecule according to any one of claims 1 to 22; 2) one or more gRNA molecules and a Cas9 molecule according to any one of claims 1 to 22. encoding nucleic acid; 3) A nucleic acid encoding one or more gRNA molecules according to any one of claims 1 to 22, and and Cas9 molecules; 4) A nucleic acid encoding one or more gRNA molecules according to any one of claims 1 to 22, and and a nucleic acid encoding a Cas9 molecule; 5) any one of 1) to 4) above and a template nucleic acid; 6) A nucleic acid comprising a sequence encoding any one of 1) to 4) above and a template nucleic acid; 7) A composition according to any one of claims 23 to 28; or ８） The vector of claim 30 [0023] A method comprising the step of contacting (e.g., introducing)

32. The cell may be an animal cell, such as a mammalian cell, a primate cell, or a human cell, such as a human optionally, the cells are cells of a hemoglobinopathy, e.g., sickle cell disease or 32. The method of claim 31, obtained from a patient suffering from thalassemia, such as β-thalassemia. How to do it.

33. The cells are HSPCs, optionally CD34+ HSPCs, optionally CD34+ CD9 33. The method of claim 31 or 32, wherein the HSPC is 0+HSPC.

34. The cells are placed in a composition comprising a population of cells enriched for CD34+ cells. The method according to any one of claims 31 to 33, wherein the

35. The cells (e.g., population of cells) can be derived from bone marrow, peripheral blood (e.g., mobilized peripheral blood), or umbilical cord blood. The method of any one of claims 31 to 34, wherein the compound is isolated from

36. The cells are autologous or allogeneic to the patient receiving the cells. The method according to any one of claims 31 to 35.

37. a) the modification is in a genomic DNA complementary to the targeting domain of the one or more gRNA molecules; introducing indels into or near the DNA sequence; and / or b) the modification comprises modifying the HBG1 promoter region of the one or more gRNA molecules complementary to the targeting domain (e.g., at least 90% complementary to the gRNA targeting domain) a sequence complementary to the gRNA targeting domain (e.g., fully complementary to the gRNA targeting domain) and the HBG2 promoter complementary to the targeting domain of the one or more gRNA molecules in the promoter region (e.g., For example, at least 90% complementary to the gRNA targeting domain, e.g., the gRNA target between the sequence (fully complementary to the target domain) and the sequence containing, e.g., substantially all of the target domain and optionally, the deletion is between 5,250,092 and 5,2 49,833, - without any nucleotides located between strands (hg38), claims 31 to 36. The method of any one of claims 36 to 36.

38. (a) a population of cells, wherein at least about 15%, for example at least about 17%, of said population %, such as at least about 20%, such as at least about 30%, such as at least about 40% , for example at least about 50%, for example at least about 55%, for example at least about 60%, For example, at least about 70%, for example at least about 75%, are modified, e.g., in and optionally, the indel is selected from the group consisting of a nucleotide sequence listed in Tables 2-7. and optionally said cells of said population are selected from among indels having an indel sequence between 5,250,092 and 5 , 249,833, - not containing a deletion of nucleotides located between the strands (hg38); (b) the modification results in cells with the ability to differentiate into differentiated cells of the erythroid lineage (e.g., red blood cells); The differentiated cells can be, for example, non-modified cells (e.g., a population of cells), exhibiting increased levels of fetal hemoglobin relative to the total number of cells; (c) the modification is in a population of differentiated cells, e.g., a population of cells of the erythroid lineage (e.g., erythroid cells) The population of differentiated cells may be, for example, An increased proportion of F cells relative to a population of unmodified cells (e.g., at least about 15% of F cells) , at least about 20%, at least about 25%, at least about 30%, or at least about 40% % higher percentage); and / or (d) the modification induces differentiation into a differentiated cell, e.g., a cell of the erythroid lineage (e.g., an erythroid cell). The differentiated cells may have a low number of phenotypes per cell, resulting in a cell (e.g., a population of cells) with the ability to differentiate. at least about 6 picograms (e.g., at least about 7 picograms, at least about 8 picograms) , at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms 38. Any of claims 31 to 37, which produces fetal hemoglobin (about 1000 mg / kg, or about 9 to about 10 picograms). The method according to any one of claims 1 to 4.

39. A cell modified by the method of any one of claims 31 to 38 or claim 31 39. A cell obtainable by the method according to any one of claims 1 to 38.

40. A cell containing an indel as described in Table 7-2, optionally comprising 5,250,092 ~5,249,833, - not including deletion of nucleotides located between strands (hg38) cell.

41. A first gRNA molecule according to any one of claims 1 to 22 or claims 23 to 28. A composition according to any one of claims 29, a nucleic acid according to claim 30, or a vector according to claim 31. -containing cells.

42. It has the ability to differentiate into differentiated cells, for example, cells of the erythroid lineage (e.g., red blood cells), modified cells are, for example, compared to cells of the same type that have not been modified to contain gRNA molecules. and optionally, the differentiated cells (e.g., red blood cells) exhibit increased levels of fetal hemoglobin. Cells of the erythroid lineage (e.g., erythroid cells) have been modified to contain gRNA molecules, e.g., at least about 6 picograms (e.g., at least about 7 picograms) of differentiated cells of the same type that are not differentiated; picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms, or about 8 to about 9 picograms, or about 9 to about 10 picograms) of fetal hemoglobin A cell according to any one of claims 39 to 41, which produces a bottle.

43. The cell of any one of claims 39 to 42, which has been contacted with a stem cell proliferation agent.

44. The stem cell proliferation agent is a) (1r,4r)-N １ -(2-benzyl-7-(2-methyl-2H-tetrazole- 5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1 ,4-diamine; b) methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5 -b] indole-7-carboxylate; c) 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H -purin-6-ylamino)ethyl)phenol; d) (S)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-( 5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; or e) Combinations of these (e.g., (1r, 4r)-N １ -(2-benzyl-7-(2- Methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indole- (S)-2-(6-(2-(1H-yl)cyclohexane-1,4-diamine and (S)-2-(6-(2-(1H-yl)cyclohexane-1,4-diamine (5-fluoropyridin-3-yl)-9H (-purin-9-yl)propan-1-ol) 44. The cell of claim 43, wherein

45. A cell, such as a cell according to any one of claims 39 to 44, a) complementary to the targeting domain of a gRNA molecule according to any one of claims 1 to 22; indels in or near genomic DNA sequences; and / or b) a gR according to any one of claims 1 to 22 in the HBG1 promoter region; complementary to the targeting domain of a gRNA molecule (e.g., at least a sequence that is 90% complementary to both the gRNA targeting domain and the gRNA targeting domain, e.g., fully complementary to the gRNA targeting domain; A gRNA molecule according to any one of claims 1 to 22 in the HBG2 promoter region complementary to the targeting domain (e.g., at least 90 % complementary, e.g., fully complementary to the gRNA targeting domain), a deletion comprising, for example, substantially all of, the sequence and optionally the deletion comprises 5,250,092 to 5,249,833, -strand (h g38), e.g., a cell according to any of claims 39 to 44, which does not contain a nucleotide located between The cell described in any one of claims 1 to 4.

46. an animal cell, such as a mammalian cell, a primate cell, or a human cell, such as a human cell optionally, hemoglobinopathies, such as sickle cell disease or thalassemia, such as β-thalassemia; Cells according to any one of claims 39 to 45, obtained from a patient suffering from racemia. Cell.

47. HSPCs, optionally CD34+ HSPCs, optionally CD34+CD90+ HSPCs The cell according to any one of claims 39 to 46,

48. The cells (e.g., population of cells) can be derived from bone marrow, peripheral blood (e.g., mobilized peripheral blood), or umbilical cord blood.

48. The cell of any one of claims 39 to 47, isolated from

49. 39 to 4, wherein the cells are autologous or allogeneic to the patient to whom they are administered.

9. A cell described in any one of 8.

50. A population of cells comprising the cells of any one of claims 39 to 49, optionally at least about 50%, such as at least about 60%, such as at least about 10%, at least about 70%, e.g., at least about 80%, e.g., at least about 90% (e.g., at least At least about 95%, at least about 96%, at least about 97%, at least about 98% or less A population of cells, at least about 99% of which are cells according to any one of claims 39 to 49. 。

51. Differentiation into a population of differentiated cells, e.g., a population of cells of the erythroid lineage (e.g., a population of red blood cells) and the population of differentiated cells is, for example, a population of unmodified cells of the same type. an increased proportion of F cells relative to the F cell population (e.g., at least about 15% of F cells, at least about 1 7%, at least about 20%, at least about 25%, at least about 30% or at least about 40% higher percentage of F cells per cell; and optionally, said F cells of said population of differentiated cells have an average of at least about 6 picograms (e.g., at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms, or from about 8 to about 9 picograms, or about 9 to about 10 picograms) of fetal hemoglobin. A population of cells according to claim 0.

52. 1) at least 1e6 CD34+ cells / kg of body weight of the patient to whom the cells are administered; 2) at least 2e6 CD34+ cells / kg of body weight of the patient to whom the cells are administered; 3) at least 3e6 CD34+ cells / kg of body weight of the patient to whom the cells are administered; 4) at least 4e6 CD34+ cells / kg of body weight of the patient to whom the cells are administered; and teeth 5) 2e6 to 10e6 CD34+ cells / kg of body weight of the patient to whom the cells are administered 52. The population of cells of claim 50 or 51, comprising:

53. At least about 40%, e.g., at least about 50%, (e.g., at least about 10%) of the cells of the population at least about 60%, at least about 70%, at least about 80%, or at least about 90%) are CD34+ cells, and optionally at least about 10% of the cells in the population, e.g. For example, at least about 15%, for example at least about 20%, for example at least about 30% 53. The population of cells according to any one of claims 50 to 52, which are D34+CD90+ cells.

54. derived from umbilical cord blood, peripheral blood (e.g., mobilized peripheral blood), or bone marrow, e.g., derived from bone marrow, 54. A population of cells according to any one of claims 50 to 53.

55. comprising, e.g., consisting of, mammalian cells, e.g., human cells, and optionally, Patients suffering from a thalassaemia, such as sickle cell disease or thalassaemia, such as β-thalassaemia 55. A population of cells according to any one of claims 50 to 54, obtained from

56. (i) the population of cells is autologous to the patient to whom it is administered, or (ii) the 56. Any of claims 50 to 55, wherein the population of cells is of allogeneic origin relative to the patient to whom it is administered. A population of cells according to claim 1.

57. A population of cells (e.g., CD34+ cells), such as the population of cells described in any one of claims 50 to 56. a population of cells comprising an indel pattern as set forth in Table 7-2, and optionally In the alternative, the indels of the indel pattern described in Table 7-2 are At least 20%, at least 30%, at least 40%, at least 50%, or a small percentage of the cells At least 60%, at least 70%, at least 80%, at least 90% or at least A population of cells (e.g., CD34+ cells), e.g., a population of cells (e.g., CD34+ cells), which are detectable in at least 95% of the cells, e.g., 57. A population of cells according to any one of paragraphs 50 to 56.

58. A composition comprising a cell or population of cells according to any one of claims 39 to 57, Optionally, in a pharmaceutically acceptable medium, e.g., a pharmaceutically acceptable medium suitable for cryopreservation. A composition comprising:

59. A method for treating hemoglobinopathies comprising administering to a subject a compound according to any one of claims 39 to 57.

59. The method of claim 58, further comprising administering to a patient a cell or population of cells of the present invention. How to do it.

60. A method for increasing fetal hemoglobin expression in a mammal, comprising the steps of: Administering to a patient the cell or population of cells described in any one of claims 58 or the composition of claim 58. providing a method for detecting a signal;

61. 61. The method of claim 60, wherein the hemoglobinopathy is β-thalassemia or sickle cell disease. How to post.

62. 1. A method of preparing cells (e.g., a population of cells), comprising: (a) a cell (e.g., a population of cells) (e.g., HSPCs (e.g., a population of HSPCs)) providing a step; (b) culturing the cells (e.g., the population of cells) in a cell culture medium containing a stem cell proliferation agent. ex vivo culturing of the (c) a first gRNA molecule according to any one of claims 1 to 22; A nucleic acid molecule encoding the first gRNA molecule according to any one of claims 23 to 28. The composition according to any one of claims 29, 29a, 29b, 29c, 29d, 29e, 29f, 29f, 29f, 30 ... Introduction into cell A method comprising:

63. After the introduction of step (c), the cells (e.g., population of cells) may be differentiated cells (e.g., , a population of differentiated cells), e.g., cells of the erythroid lineage (e.g., a population of cells of the erythroid lineage), e.g. For example, the cells have the ability to differentiate into red blood cells (e.g., a population of red blood cells), and the differentiated cells (e.g., , a population of differentiated cells) is increased relative to, for example, the same cells that have not been subjected to step (c).

63. The method of claim 62, wherein the method produces enhanced fetal hemoglobin.

64. The stem cell proliferation agent is a) (1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazole- 5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1 ,4-diamine; b) methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5 -b] indole-7-carboxylate; c) 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H -purin-6-ylamino)ethyl)phenol; d) (S)-2-(6-(2-(lH-indol-3-yl)ethylamino)-2-( 5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-l-ol; or e) Combinations thereof (e.g., (1r,4r)-N1-(2-benzyl-7-(2- Methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indole- (S)-2-(6-(2-(1H-yl)cyclohexane-1,4-diamine and (S)-2-(6-(2-(1H-yl)cyclohexane-1,4-diamine (5-fluoropyridin-3-yl)-9H -purin-9-yl)propan-1-ol) 64. The method of claim 62 or 63, wherein:

65. The cell culture medium contains thrombopoietin (Tpo), Flt3 ligand (Flt-3L), ) and human stem cell factor (SCF), and optionally, said cell culture medium comprises human interleukin (IL-1) 1 (IL-2) 2 (IL-3) 3 (IL-4) 4 (IL-5) 5 (IL-6) 6 (IL-7) 7 (IL-8) 8 (IL-9) 9 (IL-10) 10 (IL-11) 11 (IL-12) 12 (IL-13) 13 (IL-1 optionally, said cell culture medium further comprises thrombolytic enzymes such as thrombolytic enzymes (THS) and thrombolytic enzymes (THS). Tpo (Tpo), Flt3 ligand (Flt-3L), human stem cell factor (SCF) and and, if present, human IL-6 at about 10 ng / mL to about 1000 ng / mL, respectively. a range of concentrations, optionally each at a concentration of about 50 ng / mL, e.g., a concentration of 50 ng / mL 65. The method of any one of claims 62 to 64, comprising:

66. The cell culture medium may contain a stem cell proliferation agent at a concentration ranging from about 1 nM to about 1 mM, optionally about Concentrations ranging from 1 uM to about 100 nM, optionally at concentrations ranging from about 500 nM to about 750 nM a concentration of 500 nM, optionally at a concentration of about 500 nM, for example at a concentration of 500 nM or at a concentration of about 750 nM; 66. The method of any one of claims 62 to 65, for example comprising at a concentration of 750 nM.

67. The culturing in step (b) may optionally include a culturing period prior to the introducing in step (c), Alternatively, the incubation period prior to the introduction in step (c) is at least 12 hours, e.g., For example, the period is from about 1 day to about 12 days, for example, from about 1 day to about 6 days, for example, from about 1 day to about 6 days. a period of about 3 days, for example, a period of about 1 to about 2 days, for example, a period of about 2 days; 67. The method of any one of claims 62 to 66.

68. The culturing in step (b) optionally includes a culturing period after the introducing in step (c), Alternatively, the incubation period after the introduction in step (c) is at least 12 hours, e.g., For example, the period is from about 1 day to about 12 days, for example, from about 1 day to about 6 days, for example, from about 2 days to about 6 days. For a period of about 4 days, for example for a period of about 2 days, or for a period of about 3 days, or for a period of about 4 days 68. The method of any one of claims 62 to 67, wherein the period is

69. The population of cells is increased by at least three times, such as by at least four times, for example by at least five times, 69. Any one of claims 62 to 68, expanded ex vivo, for example by at least 10-fold. The method described below.

70. 70. Any of claims 62 to 69, wherein the introducing in step (c) comprises electroporation. The method according to any one of claims 1 to 4.

71. The cells (e.g., population of cells) provided in step (a) may be human cells (e.g., 71. The method of any one of claims 62 to 70, wherein the cell population is a population of human cells.

72. The cells (e.g., population of cells) provided in step (a) may be derived from bone marrow, peripheral blood (e.g., 72. The method of claim 71, wherein the cells are isolated from peripheral blood (e.g., mobilized peripheral blood) or umbilical cord blood.

73. (i) the cells (e.g., population of cells) provided in step (a) are isolated from bone marrow; isolated from the bone marrow of a patient suffering from a hemoglobinopathy, and optionally The hemoglobinopathy is sickle cell disease or thalassemia, and optionally, the thalassemia the anemia is beta thalassemia; or (ii) the cells (e.g., population of cells) provided in step (a) are isolated from peripheral blood; isolated, for example from the peripheral blood of a patient suffering from a hemoglobinopathy, and optionally wherein the hemoglobinopathy is sickle cell disease or thalassemia, and optionally the thalassemia is β thalassemia; optionally, said peripheral blood is mobilized peripheral blood, Optionally, the mobilized peripheral blood is plerioxacin, G-CSF, or a combination thereof.

73. The method of claim 72, wherein the cells are mobilized using a

74. The population of cells provided in step (a) is enriched for CD34+ cells. The method according to any one of claims 62 to 73,

75. After the introducing step (c), the cells (e.g., population of cells) are cryopreserved.

75. The method of any one of claims 62 to 74.

76. After the introducing of step (c), the cells (e.g., population of cells) are: a) in a genomic DNA sequence complementary to the targeting domain of the first gRNA molecule; an indel at or near the site; and / or b) determining the targeting domain of the first gRNA molecule in the HBG1 promoter region; complementary to the gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain, e.g., a sequence (fully complementary to the gRNA targeting domain) and a sequence (fully complementary to the HBG2 promoter region) complementary to the targeting domain of the first gRNA molecule (e.g., the gRNA target at least 90% complementary to the gRNA targeting domain, e.g., completely complementary to the gRNA targeting domain. a deletion including, for example, substantially all of, a sequence between a target sequence and a complementary sequence and optionally the indel, e.g., deletion, is between 5,250,092 and 5,249, 833, - strand (hg38) does not contain a nucleotide located between the strands of claims 62 to 75 10. The method according to any one of claims 1 to 9.

77. (a) after said introducing of step (c), at least about 40% of said cells of said population of cells , at least about 50%, at least about 60%, at least about 70%, at least about 80% , at least about 90%, at least about 95%, at least about 96%, at least about 97% , at least about 98% or at least about 99% of the targeting sequences of the first gRNA molecule. genomic DNA sequence complementary to the domain, and optionally, The indels are selected from the indels listed in Tables 2-7, and optionally, The nucleus is located between 5,250,092 and 5,249,833 - strand (hg38). Not including leotide deletion; (b) after said introducing step (c), said cells (e.g., population of cells) are of the erythroid lineage; The cells have the ability to differentiate into differentiated cells (e.g., red blood cells), and the differentiated cells are, for example, non-modified cells. exhibiting increased levels of fetal hemoglobin to the cells (e.g., population of cells); (c) after said introducing step (c), said population of cells is a population of differentiated cells, e.g., red blood cells; a population of differentiated cells (e.g., a population of red blood cells), For example, an increased proportion of F cells relative to a population of unmodified cells (e.g., a lower proportion of F cells) At least about 15%, at least about 20%, at least about 25%, at least about 30% or less at least about 40% higher; (d) after said introducing of step (c), said cells (e.g., population of cells) are differentiated cells, e.g., For example, it has the ability to differentiate into cells of the erythroid lineage (e.g., red blood cells), and , a population of differentiated cells) is at least about 6 picograms per cell (e.g., at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 1 0 picograms, or about 8 to about 9 picograms, or about 9 to about 10 picograms) of fetal hemoglobin producing robins; (e) after said introduction of step (c), off-target indels are formed in said cell. and not by, for example, next generation sequencing and / or nucleotide insertion assays. Off-target indels, as detectable by the HBG1 and / or HBG2 proteins, not be formed outside the promoter region; and / or (f) after said introduction of step (c), e.g., next generation sequencing and / or nucleofection Off-target indels as detectable by tide insertion assays, such as the HB Off-target indels outside of the G1 and / or HBG2 promoter regions are more than about 5%, such as more than about 1%, such as more than about 0.1%, such as about 0.01% of said cells of the population 77. The method of any one of claims 62 to 76, wherein the IL-14 is not detected in more than 1% of the IL-14.

78. Cells obtainable by the method according to any one of claims 62 to 77, e.g. , population of cells).

79. 79. A cell, e.g., a modified cell, e.g., the cell of claim 78, comprising: (a) at least about 40%, at least about 50%, at least about 10% of the cells in the population of cells; at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98% or less Approximately 99% of the targeting domains of the gRNA molecules of any one of claims 1 to 22 and optionally, comprising an indel in or near a genomic DNA sequence complementary to said indel. The indel is selected from the indels listed in Tables 2-7, and optionally, the cells of the population , 5,250,092 to 5,249,833, - nucleoside located between strands (hg38) Does not contain tide deletions; (b) the cells (e.g., population of cells) are differentiated cells of the erythroid lineage (e.g., erythroid cells) ) and the differentiated cells have the ability to differentiate into, for example, non-modified cells (e.g., a population of cells). and exhibiting increased levels of fetal hemoglobin; (c) the population of cells is a population of differentiated cells, e.g., a population of cells of the erythroid lineage (e.g., erythroid a population of differentiated cells, for example, a population of unmodified cells. an increased proportion of F cells relative to the F cell population (e.g., at least about 15% of F cells, at least about 2% of F cells) 0%, at least about 25%, at least about 30%, or at least about 40% higher death; (d) the cell (e.g., population of cells) is a differentiated cell, e.g., a cell of the erythroid lineage (e.g., erythroid cells), and the differentiated cells (for example, a population of differentiated cells) are At least about 6 picograms (e.g., at least about 7 picograms, at least about 8 picograms) per picograms, at least about 9 picograms, at least about 10 picograms, or from about 8 to about 9 picograms picograms, or about 9 to about 10 picograms) of fetal hemoglobin; (e) off-target indels are not formed in the cell, e.g., in the next generation Offta as detectable by sequencing and / or nucleotide insertion assays The target indel is formed outside the HBG1 and / or HBG2 promoter region. Not possible; (f) detection, for example, by next-generation sequencing and / or nucleotide insertion assays Possible off-target indels, such as the HBG1 and / or HBG2 promoters off-target indels outside the target region in more than about 5% of the cells in the population of cells; e.g., not detectable in more than about 1%, e.g., more than about 0.1%, e.g., more than about 0.01%; and / or (g) the cell or its progeny optionally comprises a gene encoding the gene of any one of claims 1 to 22. an indel in or near the genomic DNA sequence complementary to the targeting domain of the RNA molecule; When detected by detecting and optionally, the indel is detectable in a patient receiving the indel. 2-7.

79. The cell of paragraph 78.

80. an animal cell, such as a mammalian cell, a primate cell, or a human cell, such as a human cell optionally, hemoglobinopathies, such as sickle cell disease or thalassemia, such as β-thalassemia; 80. The cell of claim 78 or 79, obtained from a patient suffering from racemia.

81. HSPCs, optionally CD34+ HSPCs, optionally CD34+CD90+ HSPCs The cell according to any one of claims 78 to 80,

82. The cells (e.g., population of cells) can be derived from bone marrow, peripheral blood (e.g., mobilized peripheral blood), or umbilical cord blood.

82. The cell of any one of claims 78 to 81, isolated from

83. Claims 78-8, wherein the cells are autologous or allogeneic to the patient to whom they are administered.

3. A cell according to any one of claims 2 to 3.

84. A method for treating hemoglobinopathies, comprising the steps of: administering to a human patient a composition comprising the cell or population of cells of any one of claims 1 to 4. method.

85. A method for increasing fetal hemoglobin expression in a human patient, comprising administering to a subject a compound of claim 39-57 or 7.

84. Administering to said human patient a composition comprising the cell or population of cells according to any one of claims 8 to 83. A method comprising the steps of:

86. 85. The method of claim 84, wherein the hemoglobinopathy is β-thalassemia or sickle cell disease. How to post.

87. 3. The human patient has at least about 2e6 per kg of body weight of the human patient. 9-57 or 78-83, for example 1 kg body weight of said human patient. At least about 2e6 per claim 39-57 or 78-83 87. The method of claim 84, wherein a composition comprising CD34+ cells of method.

88. The cell or population of cells or their progeny optionally comprises any one of claims 1 to 22 to a genomic DNA sequence complementary to the targeting domain of the gRNA molecule of claim 1 or When detected by detecting indels in the vicinity, more than 16 weeks, 20 weeks after administration and optionally, the indel is detectable in said human patient for more than 24 weeks or more than 24 weeks. is selected from the indels listed in Tables 2-7; and optionally, for more than 16 weeks after administration, The inclusion of the ... The level of detection of the indel is determined by measuring the level of detection of the indel in the population of cells immediately prior to administration. 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less The method of any one of claims 84 to 87, wherein is reduced by no more than 1%.

89. A gRNA molecule according to any one of claims 1 to 22 for use as a medicament. The composition according to any one of claims 23 to 28 or 58, the nucleic acid according to claim 29, The vector according to claim 30, and the cell according to any one of claims 39 to 57 or 78 to 83 or a population of cells.

90. A gRNA molecule according to any one of claims 1 to 22 for use in the manufacture of a medicament. The composition according to any one of claims 23 to 28 or 58, the nucleic acid according to claim 29, The vector according to claim 30, the cell according to any one of claims 39 to 57 or 78 to 83 A cell or group of cells.

91. A gRNA molecule according to any one of claims 1 to 22 for use in the treatment of a disease. The composition according to any one of claims 23 to 28 or 58, the nucleic acid according to claim 29, The vector according to claim 30, the cell according to any one of claims 39 to 57 or 78 to 83 A cell or group of cells.

92. A gRNA molecule according to any one of claims 1 to 22 for use in the treatment of a disease. The composition according to any one of claims 23 to 28 or 58, the nucleic acid according to claim 29, The vector according to claim 30, the cell according to any one of claims 39 to 57 or 78 to 83 a population of cells or cells, wherein the disease is a hemoglobinopathy, and optionally The hemoglobinopathy is sickle cell disease or thalassemia (e.g., β-thalassemia) a gRNA molecule according to any one of claims 1 to 22; a gRNA molecule according to any one of claims 23 to 28 or 58 The composition according to any one of claims 29, the nucleic acid according to claim 30, the vector according to claim 31, 84. The cell or population of cells of any one of claims 39 to 57 or 78 to 83.

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