Compositions and methods for treating hemoglobinopathies

Abstract

The present invention relates to genome editing systems, reagents and methods for the treatment of hemoglobinopathies.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880020201.4 (PCT / IB2018 / 050712), filed on February 5, 2018, with the invention name “Compositions and methods for treating hemoglobinopathies”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 455,464, filed February 6, 2017, the contents of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing filed electronically in ASCII format and hereby incorporated by reference in its entirety. The ASCII copy, created on January 30, 2018, is named PAT057603-WO-PCT_SL.txt and is 258,837 bytes in size. Background Art

[0006] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria as an adaptive immune system to defend against viral attacks. When exposed to viruses, short segments of viral DNA are integrated into the CRISPR locus of the bacterial genome. RNA is transcribed from a portion of the CRISPR locus containing viral sequences. This RNA (which contains a sequence complementary to the viral genome) mediates the targeting of the Cas9 protein to sequences in the viral genome. The Cas9 protein cuts the viral target and thereby silences it.

[0007] Recently, the CRISPR / Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific single-strand breaks (SSBs) or double-strand breaks (DSBs) allows for alteration of target sequences through, for example, nonhomologous end joining (NHEJ) or homology-directed repair (HDR). Summary of the Invention

[0008] Without being bound by theory, the present invention is based in part on the discovery that CRISPR systems (e.g., a Cas9 CRISPR system, e.g., as described herein) can be used to modify cells (e.g., hematopoietic stem and progenitor cells (HSPCs)), e.g., as described herein, in a non-specific HPFH region to, e.g., increase fetal hemoglobin (HbF) expression and / or decrease beta globin (e.g., a beta globin gene having a pathogenic mutation) expression in the progeny of the modified cells (e.g., in erythroid progeny), and that the modified (e.g., modified HSPC) cells can be used to treat hemoglobinopathies, such as sickle cell disease and beta thalassemia. In one aspect, it is unexpectedly shown herein that introducing a gene editing system (e.g., a CRISPR system as described herein) into a cell (e.g., HSPC) that targets a region of the cell genome that has no known HPFH mutation or deletion profile produces a modified HSPC (e.g., an HSPC comprising one or more insertions / deletions (indels), such as those described herein), which can be effectively engrafted into an organism, persist in the implanted organism for a long time, and differentiate, including into erythrocytes with increased fetal hemoglobin expression. In addition, these modified HSPCs can be cultured ex vivo, for example, in the presence of a stem cell expander (e.g., as described herein) under conditions that allow them to expand and proliferate while maintaining stem cell properties. When a gene editing system (e.g., a CRISPR system as described herein) is introduced into HPSCs derived from patients with sickle cell disease, the modified cells and their progeny (e.g., erythroid progeny) unexpectedly show not only upregulation of fetal hemoglobin, but also a significant decrease in sickle cell beta globulin, a significant decrease in the number of sickled cells, and an increase in the number of normal red blood cells compared to unmodified cell populations.

[0009] Thus, in one aspect, the invention provides a CRISPR system (e.g., a Cas CRISPR system, e.g., a Cas9 CRISPR system, e.g., a Streptococcus pyogenes Cas9 CRISPR system) comprising one or more (e.g., one) gRNA molecules as described herein. Any gRNA molecule described herein can be used in such systems and the methods and cells described herein.

[0010] In one aspect, the invention provides a gRNA molecule comprising tracr and crRNA, wherein the crRNA comprises a targeting domain that:

[0011] a) complementary to a target sequence in a non-deleted HFPH region (e.g., a non-deleted human HPFH region);

[0012] b) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,249,833 to Chr11:5,250,237, first strand, hg38;

[0013] c) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,254,738 to Chr11:5,255,164, first strand, hg38;

[0014] d) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,250,094-5,250,237, first strand, hg38;

[0015] e) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,255,022-5,255,164, first strand, hg38;

[0016] f) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,249,833-5,249,927, -strand, hg38;

[0017] g) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,254,738-5,254,851, -strand, hg38;

[0018] h) is complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,250,139-5,250,237, first strand, hg38; or

[0019] i) combinations thereof.

[0020] In embodiments, the targeting domain comprises, for example, consists of, any one of SEQ ID NO: 1 to SEQ ID NO: 72. In embodiments, the targeting domain comprises, for example, consists of, any one 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: 28, SEQ ID NO: 34, SEQ ID 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 SEQ ID NO: 67. In an embodiment, the targeting domain comprises 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) any one of 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, for example, consisting thereof. In an embodiment, the gRNA molecule comprises a targeting domain comprising, for example, SEQ ID NO: 8, for example, consisting thereof. In an embodiment, the gRNA molecule comprises a targeting domain comprising, for example, SEQ ID NO: 67, for example, consisting thereof. In an embodiment, the gRNA molecule comprises a targeting domain comprising (for example, consisting of) a fragment of any of the above sequences.

[0021] In any of the above aspects and embodiments, the gRNA molecule may further have a region and / or characteristic as described herein. In an embodiment, the gRNA molecule includes a fragment of any of the above-mentioned targeting domains. In an embodiment, the targeting domain includes, for example, consists of, 17, 18, 19 or 20 consecutive nucleic acids of any of the listed targeting domain sequences. In an embodiment, the 17, 18, 19 or 20 consecutive nucleic acids of any of the listed targeting domain sequences are 17, 18, 19 or 20 consecutive nucleic acids arranged at the 3' end of the listed targeting domain sequence. In other embodiments, the 17, 18, 19 or 20 consecutive nucleic acids of any of the listed targeting domain sequences are 17, 18, 19 or 20 consecutive nucleic acids arranged at the 5' end of the listed targeting domain sequence. In other embodiments, the 17, 18, 19 or 20 consecutive nucleic acids of any of the listed targeting domain sequences do not include 5' or 3' nucleic acids of the listed targeting domain sequence. In embodiments, the targeting domain consists of the enumerated targeting domain sequence.

[0022] In one aspect, including any of the preceding aspects and embodiments, the crRNA portion and the tracr portion hybridize to form a marker stem comprising SEQ ID NO: 182 or 183. In one aspect, including any of the preceding aspects and embodiments, the marker stem further comprises a first marker stem extension 3' to the crRNA portion of the marker stem, wherein the first marker stem extension comprises SEQ ID NO: 184. In one aspect, including any of the preceding aspects and embodiments, the marker stem further comprises a second marker stem extension 3' to the crRNA portion of the marker stem and, if present, the first marker stem extension, wherein the second marker stem extension comprises SEQ ID NO: 185.

[0023] In one aspect, including any of the foregoing aspects and embodiments, the tracr comprises SEQ ID NO: 224 or SEQ ID NO: 225. In one aspect, including any of the foregoing aspects and embodiments, the tracr comprises SEQ ID NO: 232, optionally further comprising an additional 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides at the 3' end. In one aspect, including any of the foregoing aspects and embodiments, the crRNA comprises, 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) SEQ ID NO: 226.

[0024] In one aspect, including any of the aforementioned aspects and embodiments, the tracr comprises 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) any one of a) to j) above, further comprising at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, for example, 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, at the 3' end; l) any one of a) to k) above, further comprising 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, at the 3' end; or m) any one of a) to l) above, further comprising 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, at the 5' end (for example, at the 5' terminus).

[0025] In one aspect, including any of the foregoing aspects and embodiments, the targeting domain and the tracr are disposed on separate nucleic acid molecules. In one aspect, including any of the foregoing aspects and embodiments, the targeting domain and the tracr are disposed on separate nucleic acid molecules, and the nucleic acid molecule comprising the targeting domain comprises SEQ ID NO: 201, optionally disposed immediately 3' of the targeting domain, and the nucleic acid molecule comprising the tracr comprises, for example, consists of, SEQ ID NO: 224. In one aspect, including any of the foregoing aspects and embodiments, the crRNA portion of the marker pole comprises SEQ ID NO: 201 or SEQ ID NO: 202. In one aspect, including any of the foregoing aspects and embodiments, the tracr comprises SEQ ID NO: 187 or 188 and optionally a first tracr extension disposed 5' of SEQ ID NO: 187 or 188, if present, comprising SEQ ID NO: 189.

[0026] In one aspect, including any of the foregoing aspects and embodiments, the targeting domain and tracr are disposed on a single nucleic acid molecule, for example, wherein the tracr is disposed 3' to the targeting domain. In one aspect, the gRNA molecule comprises a loop disposed 3' to the targeting domain and 5' to the tracr. In an embodiment, the loop comprises SEQ ID NO: 186. In one aspect, including any of the foregoing aspects and embodiments, the gRNA molecule comprises, from 5' to 3', [targeting domain]-: (a) SEQ ID NO: 195; (b) SEQ ID NO: 196; (c) SEQ ID NO: 197; (d) SEQ ID NO: 198; (e) SEQ ID NO: 231; or (f) any one of (a) to (e) above, further comprising 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides at the 3' end.

[0027] In one aspect, including any preceding aspect and embodiments, the targeting domain and the tracr are arranged on a single nucleic acid molecule, and wherein the nucleic acid molecule comprises, e.g., consists of, the targeting domain and, optionally, SEQ ID NO: 231 arranged immediately 3' to the targeting domain.

[0028] In one aspect, including in any of the above aspects and embodiments, the one or optionally more than one nucleic acid molecules comprising a gRNA molecule comprises:

[0029] a) one or more, for example three, phosphorothioate modifications at the 3' end of the one or more nucleic acid molecules;

[0030] b) one or more, for example three, phosphorothioate modifications at the 5' end of the one or more nucleic acid molecules;

[0031] c) one or more, for example three, 2'-O-methyl modifications at the 3' end of the one or more nucleic acid molecules;

[0032] d) one or more, for example three, 2'-O-methyl modifications at the 5' end of the one or more nucleic acid molecules;

[0033] e) a 2'O-methyl modification at each of the 4th residue relative to the terminus, the 3rd residue relative to the terminus, and the 2nd 3' residue relative to the terminus of the one or more nucleic acid molecules;

[0034] f) a 2'O-methyl modification at each of the 4th residue from the terminus, the 3rd residue from the terminus, and the 2nd residue 5' from the terminus of the one or more nucleic acid molecules; or

[0035] f) any combination thereof.

[0036] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0037] (a) SEQ ID NO: 74;

[0038] (b) SEQ ID NO: 75; or

[0039] (c) SEQ ID NO:76.

[0040] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0041] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 77, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0042] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 77, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0043] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 78, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0044] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 78, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0045] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0046] (a) SEQ ID NO: 79;

[0047] (b) SEQ ID NO: 80; or

[0048] (c) SEQ ID NO:81.

[0049] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0050] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 82, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0051] (b) crRNA and tracr, the crRNA comprising, for example, consisting of, SEQ ID NO: 82, and the tracr comprising, for example, consisting of, SEQ ID NO: 73;

[0052] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 83, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0053] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 83, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0054] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0055] (a) SEQ ID NO: 84;

[0056] (b) SEQ ID NO: 85; or

[0057] (c) SEQ ID NO:86.

[0058] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0059] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 87, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0060] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 87, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0061] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 88, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0062] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 88, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0063] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0064] (a) SEQ ID NO: 89;

[0065] (b) SEQ ID NO: 90; or

[0066] (c) SEQ ID NO:91.

[0067] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0068] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 92, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0069] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 92, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0070] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 93, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0071] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 93, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0072] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0073] (a) SEQ ID NO: 94;

[0074] (b) SEQ ID NO: 95; or

[0075] (c) SEQ ID NO:96.

[0076] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0077] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 97, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0078] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 97, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0079] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 98, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0080] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 98, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0081] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0082] (a) SEQ ID NO: 99;

[0083] (b) SEQ ID NO: 100; or

[0084] (c) SEQ ID NO: 101.

[0085] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0086] (a) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 102, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0087] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 102, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0088] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 103, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0089] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 103, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0090] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0091] (a) SEQ ID NO: 104;

[0092] (b) SEQ ID NO: 105; or

[0093] (c) SEQ ID NO: 106.

[0094] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0095] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 107, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0096] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 107, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0097] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 108, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0098] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 108, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0099] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0100] (a) SEQ ID NO: 109;

[0101] (b) SEQ ID NO: 110; or

[0102] (c) SEQ ID NO:111.

[0103] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0104] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 112, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0105] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 112, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0106] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 113, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0107] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 113, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0108] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0109] (a) SEQ ID NO: 114;

[0110] (b) SEQ ID NO: 115; or

[0111] (c) SEQ ID NO:116.

[0112] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0113] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 117, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0114] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 117, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0115] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 118, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0116] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 118, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0117] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0118] (a) SEQ ID NO: 119;

[0119] (b) SEQ ID NO: 120; or

[0120] (c) SEQ ID NO:121.

[0121] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0122] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 122, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0123] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 122, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0124] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 123, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0125] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 123, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0126] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0127] (a) SEQ ID NO: 124;

[0128] (b) SEQ ID NO: 125; or

[0129] (c) SEQ ID NO:126.

[0130] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0131] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 127, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0132] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 127, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0133] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 128, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0134] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 128, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0135] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0136] (a) SEQ ID NO: 129;

[0137] (b) SEQ ID NO: 130; or

[0138] (c) SEQ ID NO:131.

[0139] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0140] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 132, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0141] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 132, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0142] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 133, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0143] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 133, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0144] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0145] (a) SEQ ID NO: 134;

[0146] (b) SEQ ID NO: 135; or

[0147] (c) SEQ ID NO:136.

[0148] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0149] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 137, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0150] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 137, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0151] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 138, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0152] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 138, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0153] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0154] (a) SEQ ID NO: 139;

[0155] (b) SEQ ID NO: 140; or

[0156] (c) SEQ ID NO: 141.

[0157] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0158] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 142, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0159] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 142, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0160] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 143, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0161] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 143, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0162] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0163] (a) SEQ ID NO: 144;

[0164] (b) SEQ ID NO: 145; or

[0165] (c) SEQ ID NO:146.

[0166] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0167] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 147, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0168] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 147, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0169] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 148, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0170] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 148, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0171] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0172] (a) SEQ ID NO: 149;

[0173] (b) SEQ ID NO: 150; or

[0174] (c) SEQ ID NO:151.

[0175] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0176] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 152, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0177] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 152, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0178] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 153, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0179] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 153, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0180] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0181] (a) SEQ ID NO: 154;

[0182] (b) SEQ ID NO: 155; or

[0183] (c) SEQ ID NO:156.

[0184] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0185] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 157, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0186] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 157, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0187] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 158, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0188] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 158, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0189] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0190] (a) SEQ ID NO: 159;

[0191] (b) SEQ ID NO: 160; or

[0192] (c) SEQ ID NO:161.

[0193] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0194] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 162, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0195] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 162, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0196] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 163, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0197] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 163, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0198] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0199] (a) SEQ ID NO: 164;

[0200] (b) SEQ ID NO: 165; or

[0201] (c) SEQ ID NO:166.

[0202] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0203] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 167, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0204] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 167, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0205] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 168, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0206] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 168, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0207] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0208] (a) SEQ ID NO: 169;

[0209] (b) SEQ ID NO: 170; or

[0210] (c) SEQ ID NO: 171.

[0211] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0212] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 172, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0213] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 172, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0214] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 173, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0215] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 173, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0216] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0217] (a) SEQ ID NO: 174;

[0218] (b) SEQ ID NO: 175; or

[0219] (c) SEQ ID NO:176.

[0220] In one aspect, the present invention provides a gRNA molecule comprising, for example, consisting of:

[0221] (a) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 177, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224;

[0222] (b) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 177, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73;

[0223] (c) a crRNA and a tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 178, and the tracr comprising, e.g., consisting of, SEQ ID NO: 224; or

[0224] (d) crRNA and tracr, the crRNA comprising, e.g., consisting of, SEQ ID NO: 178, and the tracr comprising, e.g., consisting of, SEQ ID NO: 73.

[0225] In one aspect, including in any of the foregoing aspects and embodiments, the present invention provides a gRNA molecule, wherein:

[0226] When a CRISPR system (e.g., an RNP as described herein) comprising the gRNA molecule is introduced into a cell, an indel is formed at or near a target sequence that is complementary to the targeting domain of the gRNA molecule; and / or

[0227] b) when a CRISPR system comprising the gRNA molecule (e.g., an RNP as described herein) is introduced into a cell, a deletion is generated comprising a sequence, for example, comprising substantially all of the sequence between a sequence in the HBG1 promoter region that is complementary to the gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain, such as completely complementary to the gRNA targeting domain) and a sequence in the HBG2 promoter region that is complementary to the gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain, such as completely complementary to the gRNA targeting domain). In embodiments, the indel does not include nucleotides in the non-deleted HPFH or transcription factor binding site.

[0228] In one aspect, including any of the above aspects and embodiments, the present invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) comprising the gRNA molecule is introduced into a cell population, at least about 15% of the cells in the population, such as at least about 17%, such as at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 70%, such as at least about 75%, an indel is formed at or near the target sequence complementary to the targeting domain of the gRNA molecule. In one aspect, including any of the above aspects and embodiments, the indel comprises at least one nucleotide of the HBG1 promoter region or at least one nucleotide of the HBG2 promoter region. In embodiments, at least about 15% of the cells in the population comprise an indel comprising at least one nucleotide of the HBG1 promoter region and an indel comprising at least one nucleotide of the HBG2 promoter region. In one aspect, including any of the foregoing aspects and embodiments, the percentage of cells comprising a population comprising an indel of at least one nucleotide in the HBG1 promoter region differs from the percentage of cells comprising a population comprising an indel of at least one nucleotide in the HBG2 promoter region by at least about 5%, such as at least about 10%, such as at least about 20%, such as at least about 30%. In embodiments, the indel is measured by next generation sequencing (NGS).

[0229] In one aspect, including in any of the above aspects and embodiments, the present invention provides a gRNA molecule, wherein when a CRISPR system comprising the gRNA molecule (e.g., an RNP as described herein) is introduced into a cell, expression of fetal hemoglobin in the cell or its progeny, e.g., its erythroid progeny, e.g., its erythroid progeny, is increased. In embodiments, when a CRISPR system comprising a gRNA molecule (e.g., an RNP as described herein) is introduced into a cell population, the percentage of F cells in the population or a population of its progeny (e.g., its erythroid progeny, e.g., its erythroid progeny) increases by at least about 15%, e.g., at least about 17%, e.g., at least about 20%, e.g., at least about 25%, e.g., at least about 30%, e.g., at least about 35%, e.g., at least about 40% (relative to the percentage of F cells in a population of cells or a population of its progeny (e.g., its erythroid progeny, e.g., its erythroid progeny) into which no gRNA molecule has been introduced). In embodiments, the cell or its progeny, e.g., erythroid progeny, e.g., erythroid progeny, produces 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 picograms to about 9 picograms, or from about 9 picograms to about 10 picograms) of fetal hemoglobin per cell.

[0230] In one aspect, including any of the preceding aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system comprising the gRNA molecule (e.g., an RNP as described herein) is introduced into a cell, no off-target indels are formed in the cell, e.g., no off-target indels are formed outside the HBG1 and / or HBG2 promoter region (e.g., within a gene, such as a coding region of a gene), e.g., detectable by next generation sequencing and / or nucleotide insertion assay.

[0231] In one aspect, including any of the preceding aspects and embodiments, the invention provides gRNA molecules, wherein when a CRISPR system comprising the gRNA molecule (e.g., an RNP as described herein) is introduced into a cell population, no off-target indels are detected in more than about 5%, such as more than about 1%, such as more than about 0.1%, such as more than about 0.01% of the cells in the cell population, e.g., no off-target indels are detected outside the HBG1 and / or HBG2 promoter region (e.g., within a gene, such as a coding region of a gene), e.g., detectable by next generation sequencing and / or nucleotide insertion assays.

[0232] In one aspect, including any of the foregoing aspects and embodiments, the cell is (or the cell population comprises) a mammalian, primate, or human cell, e.g., a human cell, e.g., the cell is (or the cell population comprises) an HSPC, e.g., an HSPC that is CD34+, e.g., an HSPC that is CD34+CD90+. In embodiments, the cell is autologous to the patient to whom the cell is to be administered. In other embodiments, the cell is allogeneic to the patient to whom the cell is to be administered.

[0233] In one aspect, the gRNA molecules, genome editing systems (e.g., CRISPR systems), and / or methods described herein relate to a cell (e.g., as described herein) that comprises or produces one or more of the following properties:

[0234] (a) 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 cells of a population of cells described herein comprise an indel at or near a genomic DNA sequence that is complementary to a targeting domain of a gRNA molecule described herein, optionally wherein the indel is selected from the indels listed in Tables 2-7, optionally wherein the cells of the population do not comprise a deletion of nucleotides arranged between 5,250,092 and 5,249,833′-strand (hg38);

[0235] (b) the cells (e.g., cell population) described herein are capable of differentiating into differentiated cells (e.g., erythrocytes) of the erythroid lineage, and wherein the differentiated cells exhibit increased levels of fetal hemoglobin, e.g., relative to unaltered cells (e.g., cell population);

[0236] (c) the cell population described herein is capable of differentiating into a differentiated cell population, e.g., an erythroid lineage cell population (e.g., a red blood cell population), and wherein the differentiated cell population has an increased percentage of F cells, e.g., at least about 15% higher, at least about 20% higher, at least about 25% higher, at least about 30% higher, or at least about 40% higher, relative to an unaltered cell population;

[0237] (d) the cells described herein are capable of differentiating into differentiated cells, e.g., cells of the erythroid lineage (e.g., erythrocytes), and wherein the differentiated cells (e.g., a population of differentiated cells) produce 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 picograms to about 9 picograms, or from about 9 picograms to about 10 picograms) of fetal hemoglobin per cell;

[0238] (e) does not form off-target indels in the cells described herein, e.g., does not form off-target indels outside the HBG1 and / or HBG2 promoter region (e.g., within a gene, e.g., the coding region of a gene), e.g., which can be detected by next generation sequencing and / or nucleotide insertion assays;

[0239] (f) no off-target indels are detected in more than about 5%, e.g., more than about 1%, e.g., more than about 0.1%, e.g., more than about 0.01% of the cells in a cell population described herein, e.g., no off-target indels are detected outside the HBG1 and / or HBG2 promoter region (e.g., within a gene, e.g., the coding region of a gene), e.g., detectable by next generation sequencing and / or nucleotide insertion assay;

[0240] (g) optionally by detecting an indel at or near a genomic DNA sequence that is complementary to the targeting domain of the gRNA molecule of any one of claims 1 to 22, the cells described herein or their progeny being detectable in the patient into which they are transplanted, e.g., detectable in the bone marrow or detectable in the peripheral blood, more than 16 weeks, more than 20 weeks, or more than 24 weeks after transplantation, optionally wherein the indel is selected from the indels listed in Tables 2 to 7, optionally wherein the indel is a large deletion-type indel;

[0241] (h) the cell population described herein is capable of differentiating into a differentiated cell population, e.g., an erythroid lineage cell population (e.g., a red blood cell population), and wherein the differentiated cell population comprises a reduced sickle cell percentage (e.g., a sickle cell percentage that is 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), e.g., relative to an unaltered cell population; and / or

[0242] (i) The cells or cell populations described herein are capable of differentiating into a differentiated cell population, e.g., an erythroid lineage cell population (e.g., a red blood cell population), and wherein the differentiated cell population includes cells that produce reduced levels of sickle hemoglobin (HbS) relative to an unaltered cell population (e.g., levels that are 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).

[0243] In one aspect, the present invention provides a composition comprising:

[0244] 1) one or more gRNA molecules described herein (including a first gRNA molecule), e.g., any of the foregoing gRNA aspects and embodiments, and a Cas9 molecule (e.g., described herein);

[0245] 2) one or more gRNA molecules described herein (including a first gRNA molecule), e.g., any of the foregoing gRNA aspects and embodiments, and a nucleic acid encoding a Cas9 molecule (e.g., described herein);

[0246] 3) nucleic acids encoding one or more gRNA molecules described herein (including a first gRNA molecule), e.g., any of the foregoing gRNA aspects and embodiments, and a Cas9 molecule (e.g., described herein);

[0247] 4) nucleic acids encoding one or more gRNA molecules described herein (including a first gRNA molecule), e.g., any of the foregoing gRNA aspects and embodiments, and nucleic acids encoding a Cas9 molecule (e.g., described herein); or

[0248] 5) any one of 1) to 4) above, and a template nucleic acid; or

[0249] 6) Any one of 1) to 4) above, and a nucleic acid comprising a sequence encoding a template nucleic acid.

[0250] In one aspect, the invention provides a composition comprising a first gRNA molecule described herein, e.g., any of the aforementioned gRNA aspects and embodiments, further comprising a Cas9 molecule, e.g., as described herein, e.g., wherein the Cas9 molecule is active or inactive Streptococcus pyogenes Cas9, e.g., wherein the Cas9 molecule comprises SEQ ID NO: 205. In some aspects, the Cas9 molecule comprises, e.g., consists of: (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.

[0251] In one aspect, including in any of the aforementioned composition aspects and embodiments, the first gRNA molecule and the Cas9 molecule are present in a ribonucleoprotein complex (RNP).

[0252] On the one hand, including in any of the aforementioned composition aspects and embodiments, the invention provides a composition further comprising a second gRNA molecule; a second gRNA molecule and a third gRNA molecule; or a second gRNA molecule, optionally a third gRNA molecule, and optionally a fourth gRNA molecule, wherein the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule are gRNA molecules as described herein, for example, the gRNA molecules of any of the aforementioned gRNA molecule aspects and embodiments, and wherein each gRNA molecule of the composition is complementary to different target sequences. In an embodiment, two or more of the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule are complementary to target sequences in the same gene or region. In an embodiment, the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule are complementary to a target sequence that is no more than 6000 nucleotides, no more than 5000 nucleotides, no more than 500 nucleotides, no more than 400 nucleotides, no more than 300 nucleotides, no more than 200 nucleotides, no more than 100 nucleotides, no more than 90 nucleotides, no more than 80 nucleotides, no more than 70 nucleotides, no more than 60 nucleotides, no more than 50 nucleotides, no more than 40 nucleotides, no more than 30 nucleotides, no more than 20 nucleotides, or no more than 10 nucleotides apart. In an embodiment, two or more of the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule include at least one gRNA molecule comprising a targeting domain complementary to a target sequence in a HBG1 promoter region, and at least one gRNA molecule comprising a targeting domain complementary to a target sequence in a HBG2 promoter region. In one aspect, including any of the aforementioned composition aspects and embodiments, the composition comprises (e.g., consists of) a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule are: (a) independently selected and targeted to a non-deleted HPFH region, e.g., as described herein, and are complementary to different target sequences; (b) independently selected from the gRNA molecules of Table 1, and are complementary to different target sequences; c) independently selected from the gRNA molecules of Table 2, and are complementary to different target sequences; or (d) independently selected from the gRNA molecules of Table 3a, and are complementary to different target sequences, (e) independently selected from the gRNA molecules of Table 3b, and are complementary to different target sequences; or (f) independently selected from the gRNA molecules of any of the aforementioned aspects and embodiments, and are complementary to different target sequences.

[0253] In one aspect, including in any of the above composition aspects and embodiments, the composition comprises a first gRNA molecule and a second gRNA molecule, wherein:

[0254] a) The first gRNA molecule is complementary to a target sequence, wherein the target sequence includes at least one nucleotide (e.g., includes 20 consecutive nucleotides) of:

[0255] i) Chr11:5,249,833 to Chr11:5,250,237 (hg38);

[0256] ii) Chr11:5,250,094-5,250,237(hg38);

[0257] iii) Chr11:5,249,833-5,249,927 (hg38); or

[0258] iv) Chr11:5,250,139-5,250,237(hg38);

[0259] b) the second gRNA molecule is complementary to a target sequence, wherein the target sequence includes at least one nucleotide (e.g., comprises 20 consecutive nucleotides) of:

[0260] i) Chr11:5,254,738 to Chr11:5,255,164 (hg38);

[0261] ii) Chr11:5,255,022-5,255,164 (hg38); or

[0262] iii) Chr11:5,254,738-5,254,851(hg38).

[0263] In one aspect, with respect to the gRNA molecule components of the composition, the composition consists of a first gRNA molecule and a second gRNA molecule.

[0264] In one aspect, including in any of the aforementioned composition aspects and embodiments, each of said gRNA molecules is in a ribonucleoprotein complex (RNP) with a Cas9 molecule, e.g., as described herein.

[0265] In one aspect, including any of the above composition aspects and embodiments, the composition comprises a template nucleic acid, wherein the template nucleic acid comprises nucleotides corresponding to nucleotides at or near the target sequence of the first gRNA. In embodiments, the template nucleic acid comprises a nucleic acid encoding: (a) human beta globulin (e.g., human beta globulin comprising one or more of the mutations G16D, E22A, and T87Q) or a fragment thereof; or (b) human gamma globulin or a fragment thereof.

[0266] In one aspect, including any of the foregoing composition aspects and embodiments, the composition is formulated in a medium suitable for electroporation.

[0267] In one aspect, including any of the aforementioned composition aspects and embodiments, each of the gRNA molecules of the composition is in an RNP with a Cas9 molecule as described herein, and wherein the concentration of each of the RNPs is less than about 10 uM, e.g., less than about 3 uM, e.g., less than about 1 uM, e.g., less than about 0.5 uM, e.g., less than about 0.3 uM, e.g., less than about 0.1 uM. In embodiments, the concentration of the RNPs is about 1 uM. In embodiments, the concentration of the RNPs is about 2 uM. In embodiments, the concentration is the concentration of the RNPs in a composition comprising cells, e.g., as described herein, optionally wherein the composition comprising cells and RNPs is suitable for electroporation.

[0268] On the one hand, the invention provides nucleic acid sequences encoding one or more gRNA molecules as described herein, for example, any of the aforementioned gRNA molecule aspects and embodiments. In an embodiment, the nucleic acid comprises a promoter operably linked to the sequence encoding one or more gRNA molecules, for example, the promoter is a promoter recognized by RNA polymerase II or RNA polymerase III, or for example, the promoter is a U6 promoter or a HI promoter.

[0269] In one aspect, including in any of the aforementioned nucleic acid aspects and embodiments, the nucleic acid further encodes a Cas9 molecule, e.g., a Cas9 molecule comprising, e.g., consisting of, any one of 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, SEQ ID NO: 242, SEQ ID NO: 243, or SEQ ID NO: 244. In embodiments, the nucleic acid comprises a promoter operably linked to the sequence encoding the Cas9 molecule, e.g., an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, a ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.

[0270] In one aspect, the invention provides a vector comprising a nucleic acid of any of the preceding nucleic acid aspects and embodiments. In embodiments, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, and an RNA vector.

[0271] In one aspect, the invention provides a method of altering a cell (e.g., a cell population) (e.g., altering the structure (e.g., sequence) of a nucleic acid at or near a target sequence within the cell), the method comprising contacting the cell (e.g., a cell population) with (e.g., introducing into the cell (e.g., a cell population)):

[0272] 1) one or more gRNA molecules described herein (e.g., any of the aforementioned gRNA molecule aspects and embodiments), and a Cas9 molecule (e.g., described herein);

[0273] 2) one or more gRNA molecules described herein (e.g., any of the foregoing gRNA molecule aspects and embodiments), and a nucleic acid encoding a Cas9 molecule (e.g., described herein);

[0274] 3) nucleic acids encoding one or more gRNA molecules described herein (e.g., any of the aforementioned gRNA molecule aspects and embodiments), and a Cas9 molecule (e.g., described herein);

[0275] 4) nucleic acids encoding one or more gRNA molecules described herein (e.g., any of the foregoing gRNA molecule aspects and embodiments), and nucleic acids encoding a Cas9 molecule (e.g., described herein);

[0276] 5) any one of 1) to 4) above, and a template nucleic acid;

[0277] 6) any one of 1) to 4) above, and a nucleic acid comprising a sequence encoding a template nucleic acid;

[0278] 7) a composition as described herein, such as the composition of any of the preceding composition aspects and embodiments; or

[0279] 8) A vector described herein, eg, a vector of any preceding vector aspect and embodiment.

[0280] In one aspect, including in any of the foregoing method aspects and embodiments, the gRNA molecule or the nucleic acid encoding the gRNA molecule and the Cas9 molecule or the nucleic acid encoding the Cas9 molecule are formulated in a single composition. In another aspect, the gRNA molecule or the nucleic acid encoding the gRNA molecule and the Cas9 molecule or the nucleic acid encoding the Cas9 molecule are formulated in more than one composition. In one aspect, more than one composition is delivered simultaneously or sequentially.

[0281] In one aspect of the methods described herein, including any of the aforementioned method aspects and embodiments, the cells are animal cells, e.g., the cells are mammalian, primate, or human cells, e.g., the cells are hematopoietic stem or progenitor cells (HSPCs) (e.g., HSPC populations), e.g., the cells are CD34+ cells, e.g., the cells are CD34+CD90+ cells. In embodiments of the methods described herein, the cells are arranged in a composition comprising a cell population enriched for CD34+ cells. In embodiments of the methods described herein, the cells (e.g., cell populations) have been isolated from bone marrow, mobilized peripheral blood, or umbilical cord blood. In embodiments of the methods described herein, the cells are autologous or allogeneic, e.g., autologous, for the patient to whom the cells are to be administered.

[0282] In one aspect of the methods described herein, including any of the aforementioned method aspects and embodiments, a) the alteration results in an insertion / deletion at or near a genomic DNA sequence that is complementary to the targeting domain of the one or more gRNA molecules; or b) the alteration results in a deletion comprising a sequence, e.g., a sequence in the HBG1 promoter region that is complementary to the one or more gRNA targeting domains (e.g., at least 90% complementary to the gRNA targeting domains, e.g., completely complementary to the gRNA targeting domains) and a sequence in the HBG2 promoter region that is complementary to the one or more gRNA targeting domains (e.g., at least 90% complementary to the gRNA targeting domains, e.g., completely complementary to the gRNA targeting domains). In aspects of the method, the insertion / deletion is an insertion or deletion of less than about 40 nucleotides, e.g., less than 30 nucleotides, e.g., less than 20 nucleotides, e.g., less than 10 nucleotides, e.g., a single nucleotide deletion.

[0283] In one aspect of the methods described herein, including any of the aforementioned method aspects and embodiments, the method produces a cell population wherein at least about 15%, e.g., at least about 17%, e.g., at least about 20%, e.g., at least about 30%, e.g., at least about 40%, e.g., at least about 50%, e.g., at least about 55%, e.g., at least about 60%, e.g., at least about 70%, e.g., at least about 75% of the population has been altered, e.g., comprises indels.

[0284] In one aspect of the methods described herein, including any of the foregoing method aspects and embodiments, the alteration produces cells (e.g., a cell population) that are capable of differentiating into differentiated cells (e.g., erythrocytes) of the erythroid lineage, and wherein the differentiated cells exhibit increased levels of fetal hemoglobin, e.g., relative to unaltered cells (e.g., a cell population).

[0285] In one aspect of the methods described herein, including any of the foregoing method aspects and embodiments, the alteration produces a cell population that is capable of differentiating into a differentiated cell population, e.g., an erythroid lineage cell population (e.g., a red blood cell population), and wherein the differentiated cell population has an increased percentage of F cells (e.g., a percentage of F cells that is at least about 15% higher, at least about 20% higher, at least about 25% higher, at least about 30% higher, or at least about 40% higher), e.g., relative to the unaltered cell population.

[0286] In one aspect of the methods described herein, including any of the foregoing method aspects and embodiments, the alteration produces a cell that is capable of differentiating into a differentiated cell, e.g., a cell of the erythroid lineage (e.g., an erythrocyte), and wherein the differentiated cell produces 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 picograms to about 9 picograms, or from about 9 picograms to about 10 picograms) of fetal hemoglobin per cell.

[0287] In one aspect, the invention provides cells altered by a method described herein, such as any of the preceding method aspects and examples.

[0288] In one aspect, the invention provides cells obtainable by a method described herein, such as any of the preceding method aspects and examples.

[0289] In one aspect, the invention provides a cell comprising a first gRNA molecule as described herein, e.g., any of the aforementioned gRNA molecule aspects or embodiments, or a composition as described herein, e.g., any of the aforementioned composition aspects or embodiments, a nucleic acid as described herein, e.g., any of the aforementioned nucleic acid aspects or embodiments, or a vector as described herein, e.g., any of the aforementioned vector aspects or embodiments.

[0290] In one aspect of the cells described herein, including in any of the aforementioned cell aspects and embodiments, the cell further comprises a Cas9 molecule, e.g., a Cas9 molecule described herein, e.g., a Cas9 molecule comprising any one of 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, SEQ ID NO: 242, SEQ ID NO: 243, or SEQ ID NO: 244.

[0291] In one aspect of the cells described herein, including any of the aforementioned cell aspects and embodiments, the cell comprises, has comprised, or will comprise a second gRNA molecule described herein, e.g., any of the aforementioned gRNA molecule aspects or embodiments, or a nucleic acid encoding said gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule comprise non-identical targeting domains.

[0292] In one aspect of the cells described herein, including any of the aforementioned cell aspects and embodiments, expression of fetal hemoglobin is increased in the cell or its progeny (e.g., its erythroid progeny, e.g., its red blood cell progeny) relative to a cell of the same cell type or its progeny that has not been modified to include the gRNA molecule.

[0293] In one aspect of the cells described herein, including any of the above cell aspects and embodiments, the cell is capable of differentiating into differentiated cells, e.g., cells of the erythroid lineage (e.g., red blood cells), and wherein the differentiated cells exhibit increased levels of fetal hemoglobin, e.g., relative to cells of the same type that have not been modified to include the gRNA molecule.

[0294] In one aspect of the cells described herein, including any of the aforementioned cell aspects and embodiments, the differentiated cell (e.g., a cell of the erythroid lineage, e.g., an erythrocyte) produces 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 picograms to about 9 picograms, or from about 9 picograms to about 10 picograms) of fetal hemoglobin, e.g., relative to a differentiated cell of the same type that has not been modified to include the gRNA molecule.

[0295] In one aspect of the cells described herein, including any of the aforementioned cell aspects and embodiments, the cells have been contacted, e.g., ex vivo, with a stem cell expander, e.g., a stem cell expander selected from the group consisting of: 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]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-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol; or e) a combination thereof (e.g., (1r,4r)-N 1In an embodiment, the stem cell expander is (S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol.

[0296] In one aspect of the cells described herein, including in any of the aforementioned cell aspects and embodiments, the cell comprises: a) an indel at or near a genomic DNA sequence that is complementary to a targeting domain of a gRNA molecule described herein, e.g., any of the aforementioned gRNA molecule aspects or embodiments; or b) a deletion comprising a sequence, e.g., a sequence in the HBG1 promoter region that is complementary to a targeting domain of a gRNA described herein (e.g., a gRNA in any of the aforementioned gRNA molecule aspects or embodiments) (e.g., at least 90% complementary to the gRNA targeting domain, e.g., completely complementary to the gRNA targeting domain) and a sequence in the HBG2 promoter region that is complementary to a targeting domain of a gRNA described herein (e.g., a gRNA in any of the aforementioned gRNA molecule aspects or embodiments) (e.g., at least 90% complementary to the gRNA targeting domain, e.g., completely complementary to the gRNA targeting domain). In one aspect, the indel is an insertion or deletion of less than about 40 nucleotides, e.g., less than 30 nucleotides, e.g., less than 20 nucleotides, e.g., less than 10 nucleotides, e.g., the indel is a single nucleotide deletion.

[0297] In one aspect of the cells described herein, including any of the above cell aspects and embodiments, the cell is an animal cell, e.g., a mammalian cell, a primate cell, or a human cell. In one aspect, the cell is a hematopoietic stem or progenitor cell (HSPC) (e.g., a HSPC population), e.g., a CD34+ cell, e.g., a CD34+CD90+ cell. In an embodiment, the cell (e.g., a cell population) has been isolated from bone marrow, mobilized peripheral blood, or umbilical cord blood. In an embodiment, the cell is autologous relative to the patient to whom the cell is to be administered. In an embodiment, the cell is allogeneic relative to the patient to whom the cell is to be administered.

[0298] On the one hand, the present invention provides cell mass as described herein, for example, including the cell mass of cell described herein (such as the cell of any above-mentioned cell aspect and embodiment).In some respects, the present invention provides cell mass, wherein at least about 50% of cell mass, for example at least about 60%, for example at least about 70%, for example at least about 80%, for example at least about 90% (such as at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%) cell is cell as described herein, for example, the cell of any above-mentioned cell aspect and embodiment.In some respects, cell mass (for example, the cell of cell mass) can be differentiated into differentiated cell mass, such as erythroid lineage cell mass (such as erythrocyte mass), and wherein the cell mass of said differentiation is for example relative to the unmodified cell mass of same type there is the F cell percentage of increase (for example, F cell percentage is high at least about 15%, high at least about 17%, high at least about 20%, high at least about 25%, high at least about 30% or high at least about 40%). In some aspects, the F cells of the differentiated cell population produce 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 picograms to about 9 picograms, or from about 9 picograms to about 10 picograms) of fetal hemoglobin per cell.

[0299] In one aspect, including any of the aforementioned cell population aspects and embodiments, the present invention provides a cell population comprising: 1) at least 1e6 CD34+ cells / kg body weight of the patient to whom the cells are administered; 2) at least 2e6 CD34+ cells / kg body weight of the patient to whom the cells are administered; 3) at least 3e6 CD34+ cells / kg body weight of the patient to whom the cells are administered; 4) at least 4e6 CD34+ cells / kg body weight of the patient to whom the cells are administered; or 5) from 2e6 to 10e6 CD34+ cells / kg body weight of the patient to whom the cells are administered. In embodiments, at least about 40%, such as at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, or at least about 90%) of the cells of the population are CD34+ cells. In embodiments, at least about 5%, such as at least about 10%, such as at least about 15%, such as at least about 20%, such as at least about 30% of the cells of the population are CD34+CD90+ cells. In embodiments, the cell population is derived from umbilical cord blood, peripheral blood (e.g., mobilized peripheral blood), or bone marrow, e.g., derived from bone marrow. In embodiments, the cell population comprises, e.g., consists of, mammary animal cells, e.g., human cells. In embodiments, the cell population is autologous with respect to the patient to whom it is to be administered. In other embodiments, the cell population is allogeneic with respect to the patient to whom it is to be administered.

[0300] In one aspect, the present invention provides a composition comprising a cell as described herein, e.g., a cell of any of the aforementioned cell aspects and embodiments, or a cell population as described herein, e.g., a cell population of any of the aforementioned cell population aspects and embodiments. In one aspect, the composition comprises a pharmaceutically acceptable medium, e.g., a pharmaceutically acceptable medium suitable for cryopreservation.

[0301] In one aspect, the invention provides a method of treating a hemoglobinopathy comprising administering to a patient a cell described herein (e.g., a cell of any of the preceding cell aspects and embodiments), a cell population described herein (e.g., a cell population of any of the preceding cell population aspects and embodiments), or a composition described herein (e.g., a composition of any of the preceding composition aspects and embodiments).

[0302] In one aspect, the invention provides a method of increasing fetal hemoglobin expression in a mammal, the method comprising administering to the patient a cell described herein (e.g., a cell of any of the aforementioned cell aspects and embodiments), a cell population described herein (e.g., a cell population of any of the aforementioned cell population aspects and embodiments), or a composition described herein (e.g., a composition of any of the aforementioned composition aspects and embodiments). In some aspects, the hemoglobinopathy is beta-thalassemia. In some aspects, the hemoglobinopathy is sickle cell disease.

[0303] In one aspect, the present invention provides a method of preparing a cell (e.g., a cell population), the method comprising:

[0304] (a) providing a cell (e.g., a cell population) (e.g., an HSPC (e.g., an HSPC population));

[0305] (b) culturing the cells (e.g., the cell population) ex vivo in a cell culture medium comprising a stem cell expander; and

[0306] (c) introducing into the cell a first gRNA molecule, e.g., a first gRNA molecule as described herein, e.g., a first gRNA molecule of any of the aforementioned gRNA molecule aspects and embodiments; a nucleic acid molecule encoding the first gRNA molecule; a composition as described herein, e.g., a composition of any of the aforementioned composition aspects and embodiments; or a vector as described herein, e.g., a vector of any of the aforementioned aspects and embodiments. In various aspects of the method, after the introduction of step (c), the cell (e.g., a cell population) is capable of differentiating into differentiated cells (e.g., a differentiated cell population), e.g., erythroid lineage cells (e.g., an erythroid lineage cell population), e.g., red blood cells (e.g., an erythroid cell population), and wherein the differentiated cells (e.g., a differentiated cell population) produce increased fetal hemoglobin, e.g., relative to the same cells not subjected to step (c). In aspects of the method, the stem cell expander is: a) (1r,4r)-N1-(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]indole-7-carboxylate; c) 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; d) (S)-2-(6- (S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol; or (S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol. In an embodiment, the stem cell expander is (S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol. In some aspects, cell culture medium includes thrombopoietin (Tpo), Flt3 ligand (Flt-3L) and human stem cell factor (SCF). In some aspects, cell culture medium also includes human interleukin-6 (IL-6). In some aspects, cell culture medium includes each concentration range from about 10ng / mL to about 1000ng / mL, for example, each concentration is about 50ng / mL, for example, each concentration is thrombopoietin (Tpo), Flt3 ligand (Flt-3L) and human stem cell factor (SCF) of 50ng / mL. In some aspects, cell culture medium includes each concentration range from about 10ng / mL to about 1000ng / mL, for example, each concentration is about 50ng / mL, for example, each concentration is human interleukin-6 (IL-6) of 50ng / mL.In some aspects, the cell culture medium comprises a stem cell expander at a concentration ranging from about 1 nM to about 1 mM, for example, a stem cell expander at a concentration ranging from about 1 uM to about 100 nM, for example, a stem cell expander at a concentration ranging from about 500 nM to about 750 nM. In some aspects, the cell culture medium comprises a stem cell expander at a concentration of about 500 nM, for example, a stem cell expander at a concentration of 500 nM. In some aspects, the cell culture medium comprises a stem cell expander at a concentration of about 750 nM, for example, a stem cell expander at a concentration of 750 nM.

[0307] In aspects of the method for preparing cells (e.g., a cell population), the culturing of step (b) includes a culturing period prior to the introduction of step (c), for example, the culturing period prior to the introduction of step (c) is at least 12 hours, for example, a period of about 1 day to about 12 days, for example, a period of about 1 day to about 6 days, for example, a period of about 1 day to about 3 days, for example, a period of about 1 day to about 2 days, or a period of about 2 days. In aspects of the method for preparing cells (e.g., a cell population), including any of the foregoing aspects and embodiments of the method, the culturing of step (b) includes a culturing period after the introduction of step (c), for example, the culturing period after the introduction of step (c) is at least 12 hours, for example, a period of about 1 day to about 12 days, for example, a period of about 1 day to about 6 days, for example, a period of about 2 days to about 4 days, for example, a period of about 2 days or a period of about 3 days or a period of about 4 days. In aspects of the method of preparing cells (e.g., a cell population), including any of the aforementioned aspects and embodiments of the method, the cell population is expanded, e.g., at least 4-fold, e.g., at least 5-fold, e.g., at least 10-fold relative to cells not cultured according to step (b).

[0308] In the aspect of the method for preparing cell (for example, cell mass), be included in any aforementioned aspect and embodiment of the method, the introducing of step (c) comprises electroporation.In some respects, electroporation comprises 1 to 5 pulses, for example 1 pulse, and wherein each pulse is in scope under the pulse voltage of 700 volts to 2000 volts, and has the pulse duration of scope from 10ms to 100ms.In some respects, electroporation comprises 1 pulse and is for example made up of 1 pulse.In some respects, pulse (or more than one pulse) voltage range is 1500 to 1900 volts, for example 1700 volts.In some respects, the pulse duration of a pulse or more than one pulse is in the scope of 10ms to 40ms, for example, is 20ms.

[0309] In the aspect of the method for preparing cells (e.g., cell mass), including any of the aforementioned aspects and embodiments of the method, the cells (e.g., cell mass) provided in step (a) are human cells (e.g., human cell mass). In the aspect of the method for preparing cells (e.g., cell mass), including any of the aforementioned aspects and embodiments of the method, the cells (e.g., cell mass) provided in step (a) are separated from bone marrow, peripheral blood (e.g., mobilized peripheral blood) or umbilical cord blood. In the aspect of the method for preparing cells (e.g., cell mass), including any of the aforementioned aspects and embodiments of the method, the cells (e.g., cell mass) provided in step (a) are separated from bone marrow, e.g., from the bone marrow of a patient suffering from a hemoglobinopathy.

[0310] In the method of preparing a cell (eg, a cell population), including in any of the foregoing aspects and embodiments of the method, the cell population provided in step (a) is enriched for CD34+ cells.

[0311] In aspects of the method of preparing cells (eg, a cell population), including in any of the foregoing aspects and embodiments of the method, subsequent to the introduction of step (c), the cells (eg, a cell population) are cryopreserved.

[0312] In aspects of the method of preparing a cell (e.g., a cell population), including any of the foregoing aspects and embodiments of the method, following the introduction of step (c), the cell (e.g., cell population) comprises: a) an insertion / deletion at or near a genomic DNA sequence that is complementary to the targeting domain of the first gRNA molecule; or b) a deletion comprising a sequence, e.g., a sequence in the HBG1 promoter region that is complementary to the first gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain, e.g., fully complementary to the gRNA targeting domain) and a sequence in the HBG2 promoter region that is complementary to the first gRNA targeting domain (e.g., at least 90% complementary to the gRNA targeting domain, e.g., fully complementary to the gRNA targeting domain).

[0313] In a method of preparing a cell (e.g., a cell population), including any of the aforementioned aspects and embodiments of the method, after introducing 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 cells in the cell population comprise an indel at or near a genomic DNA sequence that is complementary to the targeting domain of the first gRNA molecule.

[0314] In one aspect, the invention provides cells (eg, a cell population) obtainable by a method of making a cell (eg, a cell population) described herein (eg, as described in any of the above method of making a cell aspects and examples).

[0315] In one aspect, the present invention provides a method of treating a hemoglobinopathy in a human patient, the method comprising administering to the human patient a composition comprising a cell described herein (e.g., a cell of any of the foregoing cell aspects and embodiments); or a cell population described herein (e.g., a cell population of any of the foregoing cell population aspects and embodiments). In some aspects, the hemoglobinopathy is beta-thalassemia. In some aspects, the hemoglobinopathy is sickle cell disease.

[0316] In one aspect, the present invention provides a method of increasing fetal hemoglobin expression in a human patient, the method comprising administering to the human patient a composition comprising a cell described herein (e.g., a cell of any of the foregoing cell aspects and embodiments); or a cell population described herein (e.g., a cell population of any of the foregoing cell population aspects and embodiments). In some aspects, the human patient has beta-thalassemia. In some aspects, the human patient has sickle cell disease.

[0317] In aspects of methods for treating hemoglobinopathies or methods for increasing expression of fetal hemoglobin, a composition comprising at least about 1e6 cells (e.g., cells as described herein) per kilogram of human patient body weight, e.g., at least about 1e6 CD34+ cells (e.g., cells as described herein) per kilogram of human patient body weight is administered to a human patient. In aspects of methods for treating hemoglobinopathies or methods for increasing expression of fetal hemoglobin, a composition comprising at least about 2e6 cells (e.g., cells as described herein) per kilogram of human patient body weight, e.g., at least about 2e6 CD34+ cells (e.g., cells as described herein) per kilogram of human patient body weight is administered to a human patient. In aspects of methods for treating hemoglobinopathies or methods for increasing expression of fetal hemoglobin, a composition comprising about 2e6 cells (e.g., cells as described herein) per kilogram of human patient body weight, e.g., about 2e6 CD34+ cells (e.g., cells as described herein) per kilogram of human patient body weight is administered to a human patient. In aspects of methods of treating hemoglobinopathies or methods of increasing fetal hemoglobin expression, a composition comprising at least about 3e6 cells (e.g., cells as described herein) per kilogram of human patient body weight, e.g., at least about 3e6 CD34+ cells (e.g., cells as described herein) per kilogram of human patient body weight is administered to a human patient. In aspects of methods of treating hemoglobinopathies or methods of increasing fetal hemoglobin expression, a composition comprising about 3e6 cells (e.g., cells as described herein) per kilogram of human patient body weight, e.g., about 3e6 CD34+ cells (e.g., cells as described herein) per kilogram of human patient body weight is administered to a human patient. In aspects of methods of treating hemoglobinopathies or methods of increasing fetal hemoglobin expression, a composition comprising about 2e6 to about 10e6 cells (e.g., cells as described herein) per kilogram of human patient body weight, e.g., about 2e6 to about 10e6 CD34+ cells (e.g., cells as described herein) per kilogram of human patient body weight is administered to a human patient.

[0318] In one aspect, the invention provides: a gRNA molecule described herein, such as a gRNA molecule of any of the aforementioned gRNA molecule aspects and embodiments; a composition described herein, such as a composition of any of the aforementioned composition aspects and embodiments, a nucleic acid described herein, such as a nucleic acid of any of the aforementioned nucleic acid aspects and embodiments; a vector described herein, such as a vector of any of the aforementioned vector aspects and embodiments; a cell described herein, such as a cell of any of the aforementioned cell aspects and embodiments; or a cell population described herein (such as a cell population of any of the aforementioned cell population aspects and embodiments) for use as a medicament.

[0319] In one aspect, the invention provides: a gRNA molecule as described herein, such as a gRNA molecule of any of the aforementioned gRNA molecule aspects and embodiments; a composition as described herein, such as a composition of any of the aforementioned composition aspects and embodiments, a nucleic acid as described herein, such as a nucleic acid of any of the aforementioned nucleic acid aspects and embodiments; a vector as described herein, such as a vector of any of the aforementioned vector aspects and embodiments; a cell as described herein, such as a cell of any of the aforementioned cell aspects and embodiments; or a cell population as described herein (such as a cell population of any of the aforementioned cell population aspects and embodiments), for use in the preparation of a medicament.

[0320] In one aspect, the invention provides: a gRNA molecule as described herein, such as a gRNA molecule of any of the aforementioned gRNA molecule aspects and embodiments; a composition as described herein, such as a composition of any of the aforementioned composition aspects and embodiments, a nucleic acid as described herein, such as a nucleic acid of any of the aforementioned nucleic acid aspects and embodiments; a vector as described herein, such as a vector of any of the aforementioned vector aspects and embodiments; a cell as described herein, such as a cell of any of the aforementioned cell aspects and embodiments; or a cell population as described herein (such as a cell population of any of the aforementioned cell population aspects and embodiments) for use in treating a disease.

[0321] In one aspect, the invention provides: a gRNA molecule described herein, such as a gRNA molecule of any of the aforementioned gRNA molecule aspects and embodiments; a composition described herein, such as a composition of any of the aforementioned composition aspects and embodiments, a nucleic acid described herein, such as a nucleic acid of any of the aforementioned nucleic acid aspects and embodiments; a vector described herein, such as a vector of any of the aforementioned vector aspects and embodiments; a cell described herein, such as a cell of any of the aforementioned cell aspects and embodiments; or a cell population described herein (such as a cell population of any of the aforementioned cell population aspects and embodiments), for use in treating a disease, wherein the disease is a hemoglobinopathy, such as β-thalassemia or sickle cell disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0322] Figure 1 : HbF induction was performed 7 days after editing. For each gRNA target sequence tested, the percentage of cells with induced HbF expression based on mock transfection, corrected for background levels, is shown as the mean, with error bars indicating standard deviation. gRNA G8, targeting exon 2 of BCL11A, was used as a positive control. The dashed line of 17% represents the threshold level selected for analysis. As shown in the legend, various shades of gray correlate the extent of HbF induction with the extent of editing in the HBG1 or HBG2 target loci.

[0323] Figure 2: Editing efficiency of the HBG1 target locus. For each gRNA tested, the percentage of indels detected by NGS is shown as the mean, with error bars representing standard deviation. gRNA G8 targeting exon 2 of BCL11A was used as a positive control. Arrows indicate two guides for which no NGS data were obtained.

[0324] Figure 3 : Editing efficiency of the HBG2 target locus. For each gRNA tested, the percentage of indels detected by NGS is shown as the mean, with error bars indicating standard deviation. gRNA G8 targeting exon 2 of BCL11A was used as a positive control. Arrows indicate the sixteen guides for which no NGS data were obtained.

[0325] Figure 4 : Overview of the locations of high-performing gRNA target sequences (e.g., >17% HbF upregulation at day 7), known non-deletion 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.

[0326] Figure 5 : Overview of the locations of high-performing gRNA target sequences (e.g., >17% HbF upregulation at day 7), known non-deletion 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.

[0327] Figure 6 Figure 3: Editing efficiency of different Cas9 variants at the targeted B2M locus in CD34+ HSPCs assessed by NGS and flow cytometry. NLS = SV40 NLS; His6 (SEQ ID NO: 247) or His8 (SEQ ID NO: 248) refers to 6 (SEQ ID NO: 247) or 8 (SEQ ID NO: 248) histidine residues, respectively; TEV = tobacco etch virus cleavage site; Cas9 = wild-type Streptococcus pyogenes Cas9 - mutation or variant as indicated).

[0328] Figure 7 : Detection and quantification of HbF-positive cells by flow cytometry at 7 days (black columns), 14 days (light gray columns), or 21 days (dark gray columns) after erythroid differentiation after electroporation of HSPCs with RNPs containing sgRNAs targeting the specified domains. The percentage of HbF+ cells in control cultures not treated with sgRNA at each time point has been subtracted. Mean values ​​+ standard deviations are indicated (n = 2 technical replicates).

[0329] Figure 8: After electroporation of HSPCs with RNPs containing sgRNAs of the specified targeting domains, detection and quantification of HbF-positive cells by flow cytometry at 7 days (hollow black columns), 14 days (hollow light gray columns), or 21 days (hollow dark gray columns) after erythroid differentiation. The percentage of HbF+ cells in control cultures not treated with sgRNA at each time point has been subtracted. The average value (column) of two independent cell donors is shown, as well as the value of each donor (circle = first donor, triangle = second donor).

[0330] Figure 9 . PCR products were visualized from the indicated reactions: P1, P2, or P3 as described in the Examples from cells electroporated with RNPs containing 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 alleles) or 4.9 kb (for inversion alleles), 2.8 kb (for 4.9 kb deletion alleles). P2: 3.8 kb (for wild-type / small indel alleles), no product (for 4.9 kb deletion or 4.9 kb inversion alleles). P3: 1.8 kb (for 4.9 kb inversion alleles), no product (for wild-type / small indel or 4.9 kb deletion alleles). L = DNA reference ladder. * = DNA was isolated from this band and subjected to next-generation sequencing.

[0331] Figure 10 Schematic diagram indicating the genomic locations of primer and probe binding sites used for a digital droplet PCR assay to quantify a 4.9 kb deletion. Primers (5.2 kb forward and 5.2 kb reverse) 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 this region is not bound by the primers. The regions in the HBG1 and HBG2 promoters where the targeting domains are located 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.

[0332] Figure 11:Sorting scheme of HSPC subpopulation of cell sample after electroporation with RNP containing sgRNA of GCR-0067 targeting domain. Shown is the dot plot of cell fluorescence after immunostaining targeting specified cell surface markers. The following groups are sorted as follows: P5=CMP (CD34+CD45RA-CD38+), P9=MPP (CD34+CD45RA-CD38-CD90-CD49f-), P10=ST-HSC (CD34+CD45RA-CD38-CD90-CD49f+), P11=LT-HSC (CD34+CD45RA-CD38-CD90+CD49f+). Total CD34+ cells (not shown) are also sorted.

[0333] Figure 12 : The editing percentage of HSPC subpopulations sorted after electroporation with RNPs containing sgRNA targeting the GCR-0067 domain. HBG1 indels and HBG2 indels represent the percentage of small insertions and deletions identified by next-generation sequencing of PCR amplicons at or near the HBG1 or HBG2 promoter region targeting domain, respectively (note that alleles with the previously described 4.9 kb deletion or inversion were not amplified). HBG1-HBG2 deletions indicate the percentage of alleles with 4.9 kb deletions, as determined by the digital droplet PCR assay described in the example. Total editing is an approximate value calculated by adding the percentage of HBG1-HBG2 deletions to the percentage without HBG1-HBG2 deletions multiplied by the HBG2 indel percentage.

[0334] Figure 13: Figure 13A Shown is the sum of all indels observed at the HBG1 locus in the cell type after introduction of an sgRNA containing the targeting domain of GCR-0067. Indels are arranged with the most common indels at the top of each column. Not quantified in this assay is the fraction of cells containing a large 4.9 kb deletion between the HBG1 and HBG2 loci. The number in each column of the most common indel represents the number of nucleotide differences from the wild-type genomic sequence (- for deletions; + for insertions). Figure 13BThe sum of all insertions / deletions observed at the HBG2 locus in the cell type after the introduction of an sgRNA comprising a targeting domain of GCR-0067 is shown. Insertions / deletions are arranged with the most common insertions / deletions at the top of each column. What was not quantified in this assay was the fraction of cells containing a large 4.9kb deletion between the HBG1 and HBG2 loci. The numbers in each column of the most prevalent insertion / deletion represent the number of nucleotide differences from the wild-type genomic sequence (- for deletion; + for 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.

[0335] Figure 14 : Percentage of colonies corresponding to the indicated subtypes CFU-GEMM (dark grey bars), CFU-G / M / GM (light grey bars), or BFU-E / CFU-E (light grey bars) after electroporation with RNPs containing sgRNAs targeting the indicated domains or control cultures not treated with sgRNAs. Mean values ​​+ / - standard deviations are indicated (n = 2 independent donors).

[0336] Figure 15 : Multiplication of total nucleated cells (TNC), CD34 positive cells (CD34+), and CD34 and CD90 double positive cells (CD34+CD90+) as shown in culture medium containing compound 4 after electroporation with RNPs containing sgRNAs targeting the GCR-0067 domain or control cultures not treated with sgRNA. Mean values ​​+ / - standard deviations are indicated (n = 2 independent donors). The mean values ​​(bars) of three independent cell donors are shown, as well as the values ​​for each donor (square = donor A, triangle = donor B, circle = donor C). The differences between edited and control cultures were not significant (ns) by unpaired t-test (GraphPad Prism).

[0337] Figure 16 : Representative gating of cell populations by flow cytometry. Dot plots of cell fluorescence after immunostaining targeting specified cell surface markers or isotype controls (isotypes) are shown. The gates indicated by thick boxes are used to quantify the percentage of the specified population and are set to exclude cells labeled with isotype controls. Only live cells pre-gated as DAPI negative and within the cell forward scatter and side scatter gates are shown, and these cells are derived from donor C electroporated only with Cas9.

[0338] Figure 17: Percentage of cells with the indicated cell surface phenotypes as assessed by flow cytometry after electroporation of RNPs containing sgRNA targeting the GCR-0067 domain (black bars) or control cultures not treated with sgRNA (grey bars). Cells were expanded 2 days after electroporation of RNPs in medium containing compound 4 and analyzed by Figure 16 Flow cytometry evaluation as described in . Mean + standard deviation (n = 3 independent donors) are indicated. There were no significant differences between edited and unedited cells for a given population by unpaired t-test (GraphPad Prism).

[0339] Figure 18A CE-MS quantification of globin subunits in mock-edited HSCs derived from a sickle cell disease patient (SCD1) using Cas9 and no sgRNA. Following differentiation into the erythroid lineage, the cells displayed normal levels of α-globin, no normal β-globin (due to sickle homozygosity), high levels of the sickle β-globin subunit, and low levels of fetal γ-globin.

[0340] Figure 18B CE-MS quantification of globin subunits in genome-edited HSPCs derived from a sickle cell patient (SCD1). Following HSC editing, erythroid cells derived from the sample patient exhibited a 40% upregulation of fetal γ-globin and a concomitant 50% downregulation of sickle β-globin subunits.

[0341] Figure 19 Schematic scheme of cell implantation for studying gene editing. Transplantation of HSCs gene-edited with Cas9 and an sgRNA containing the targeting domain of CR001128 (sg1128). Half a million human CD34+ cells were edited with either the gRNA or the gene-edited sg1128 and then injected into 2 Gy-irradiated NOD.Cg-Prkdc scid Il2rg tm1Wjl Mice were bled at 4, 8, 12, and 16 weeks, and bone marrow cells were harvested 16 weeks after transplantation.

[0342] Figure 20 .use Figure 19 The experimental protocol shown in Figure 1 was for bone marrow reconstitution 16 weeks after transplantation.

[0343] Figure 21 .use Figure 19 Reconstitution of myeloid, B-lymphoid, and T-lymphoid cells in peripheral blood and bone marrow at multiple time points according to the experimental protocol shown in . N = 5 / group, data shown minimum to maximum, 4 independent experiments.

[0344] Figure 22 Schematic diagram of transplantation studies evaluating the 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 to a gRNA from the erythroid-specific enhancer region of the BCL11A gene (sg-G1128; also referred to as sg1128). Cells were left in culture for 24 hours after electroporation. Culture conditions before and after electroporation included StemSpan SFEM plus IL6, SCF, TPO, and Flt3L; and 750 nM Compound 4.

[0345] Figure 23 : Artificial implantation in NSG mice and lineage analysis over 20 weeks. Figure 23 A) Peripheral blood chimerism after 18 weeks; Figure 23 B) Lineage distribution in peripheral blood at 18 weeks. Bone marrow analysis: Figure 23 C) Bone marrow analysis of human cells at week 9; Figure 23 D) Human CD45+ engraftment and lineage distribution of human cells in the bone marrow at week 9; Figure 23 E) Human CD45+ engraftment in bone marrow at week 20 post-implantation; Figure 23 F) Lineage distribution of engrafted cells in the bone marrow at 20 weeks.

[0346] Figure 24A 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 (sg1128) from the erythroid-specific enhancer region of the BCL11A gene. Human cell engraftment in NSG mice at 8 weeks post-transplantation is shown. N = 10 / group, 3 independent experiments. Graphs show pooled data.

[0347] Figure 24B 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 (sg1128) from the erythroid-specific enhancer region of the BCL11A gene. Human cell engraftment in NSG mice at 20 weeks post-transplantation is shown. N = 10 / group, 3 independent experiments. Graphs show pooled data.

[0348] Figure 25 Multilineage reconstitution in NSG mice transplanted with gene-edited CD34+ cells. N = 10 / group, data from one representative experiment.

[0349] Figure 26 .Maintain high editing efficiency before and after transplantation. “Pre-Xpt”: Editing efficiency and indel patterns of individual sgRNAs in human CD34+ cells measured by NGS after editing but before transplantation. “8 weeks after Xpt”: Editing efficiency and indel patterns of human CD34+ cells 8 weeks after mouse bone marrow transplantation measured by NGS. “20 weeks after Xpt”: Editing efficiency and indel patterns of human CD34+ cells 20 weeks after mouse bone marrow transplantation measured by NGS. Each group was electroporated in triplicate, and data are from one representative experiment. As used with respect to this figure, “indels” refers to the sum of all indels smaller than 200nt; “large deletions” refers to deletions of the sequence between the predicted HBG1 and HBG2 binding sites for each gRNA.

[0350] Figure 27 NGS analysis of CD34+ cells after RNP editing. sgRNAs targeting specific regions are indicated on the x-axis. Figure 27 A: NGS analysis of CD34+ cells on day 2 after RNP electroporation. Figure 27 B: NGS analysis of whole bone marrow of NSG mice transplanted with edited bone marrow CD34+ cells at 9 weeks post-transplantation (27B). Figure 27 C: NGS analysis of whole bone marrow from NSG mice transplanted with edited bone marrow CD34+ cells at week 20 post-transplant. Insertional indels are indicated in black, and deletional indels (excluding large deletions, which include excision of the region between the binding HBG1 and HBG2 target sequences for each of gRNAs sg-G51, sg-G48, and sg-G67) are indicated in gray. The total editing percentage is indicated by the height of the bar. N = 10, data are presented as the mean from one independent experiment. + SEM.

[0351] Figure 28 Gene-edited, long-term engrafted human HSCs are able to produce increased levels of HbF following erythroid differentiation. Fifty thousand human CD34+ cells were sorted from the bone marrow of 8- or 20-week-old transplanted NSG mice. The sorted cells were inoculated into erythroid differentiation medium for 14-21 days. Mature erythrocytes in culture were assayed for HbF expression and the number of HbF+ cells was counted by flow cytometry. Mock controls represent CD34+ cells edited with Cas9 in the absence of gRNA and transplanted into NSG mice in the same manner as the gene-edited control group. N = 10 / group, 3 independent experiments.

[0352] Figure 29The off-target activity of HBG1 and / or HBG2 guide RNAs was assessed using a dsDNA oligonucleotide insertion assay in HEK-293 cells overexpressing Cas9. The target site (open circles) and potential off-target sites detected (filled circles) are indicated; the y-axis represents the frequency of detection. All gRNAs were tested in dgRNA format, with the targeting domain indicated by the CRxxxxxx identifier.

[0353] Figure 30 The off-target activity of HBG1 and / or HBG2 guide RNAs was assessed using a dsDNA oligonucleotide insertion assay in HEK-293 cells overexpressing Cas9. The target site (open circles) and potential off-target sites detected (filled circles) are indicated; the y-axis indicates the frequency of detection. gRNAs were tested in either dgRNA format (where the targeting domain is indicated by a CRxxxxx identifier) ​​or sgRNA format (where the targeting domain is indicated by a Gxxxxxx identifier).

[0354] Figure 31A CD34+ cell counts of cells derived from mobilized peripheral blood of healthy individuals after gene editing. Cells were thawed on day 0 and cultured for 3 days before electroporation on day 3. CD34+ cell counts were counted by ISHAGE 10 days after electroporation. Two independent experiments were performed in duplicate. Total N = 5. The graph shows data from one experiment (mean + SEM).

[0355] Figure 31B Expansion of CD34+ cells derived from mobilized peripheral blood of healthy individuals after gene editing. Cells were thawed on day 0 and cultured for 3 days before electroporation on day 3. Expansion of total monocytes on days 3, 7, and 10 after editing. Two independent experiments were performed in duplicate. Total N = 5. Graphs show data from one experiment (mean values). + SEM).

[0356] Figure 31C CD34+ cell viability of cells derived from mobilized peripheral blood of healthy individuals after gene editing. Cells were thawed on day 0 and cultured for 3 days before electroporation on day 3. Viability of monocytes at the same time point after editing. Two independent experiments were performed in duplicate. Total N = 5. Graphs show data from one experiment (mean + SEM).

[0357] Figure 32AEditing efficiency of sg1128 and sg0067 in CD34+ cells mobilized from peripheral blood of healthy individuals. Shown are the percentages of indels captured by NGS after editing using sg1128 (targeting the BCL11A+58 region of ESH) and sg0067 (targeting the HbG-1 and HbG-2 gene clusters). The figure also indicates the overall editing efficiency of sg1128, as this sgRNA only produced small indels. Five or more independent experiments were performed in duplicate or triplicate, n = 2-3 / experiment.

[0358] Figure 32B Editing efficiency of sg1128 and sg0067 in CD34+ cells mobilized from peripheral blood of healthy individuals. A. Percentage of indels captured by NGS after editing with sg0067 (targeting the HbG-1 and HbG-2 gene clusters). Shown are the overall editing efficiency and editing pattern of sg0067. The editing pattern of sg0067 consists of large 5 kb deletions (indicated by black bars) and smaller indels (indicated by gray bars). Five or more independent experiments were performed in duplicate or triplicate, n = 2-3 / experiment.

[0359] Figure 33 .Fold change of gamma-globin transcripts in erythroid cells from patient samples after CRISPR knockdown of BCL11A or indel formation in the HBG1 / 2 region. CD34+ cells derived from mobilized peripheral blood of healthy donors were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the previous procedure. On day 11 of erythroid differentiation, cells were harvested from culture and qPCR was performed to measure gamma-globin and beta-globin transcripts, normalized to GAPDH. Experiments were performed twice independently, each in duplicate, n=2-3 / experiment. Data show the average of the pooled donors from one study. + SEM.

[0360] Figure 34 . Counting HbF+ cells from healthy individuals after gene editing. CD34+ cells derived from mobilized peripheral blood of healthy donors were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the previous procedure. On day 10 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody to count HbF+ cells by flow cytometry. The experiment was performed twice independently, each time in duplicate, n=2-3 / experiment. Data show the average value from one study + SD.

[0361] Figure 35A. Expansion and viability of CD34+ cells derived from peripheral blood of individuals with sickle cell disease after gene editing. Cells were cultured for 6-10 days before electroporation. D0 refers to the day of electroporation. Absolute counts of CD34+ cells by ISHAGE 10 days after electroporation are shown. N = 4, data show mean values. + SEM. Four independent experiments were performed in duplicate. Bars at each time point are mock, sg1128, and sg0067 from left to right.

[0362] Figure 35 B. Expansion and viability of CD34+ cells derived from peripheral blood of individuals with sickle cell disease after gene editing. Cells were cultured for 6-10 days before electroporation. D0 refers to the day of electroporation. The percentage of CD34+ cells after ISHAGE 10 days after electroporation is shown. N = 4, data show mean values. + SEM. Four independent experiments were performed in duplicate. Bars at each time point are mock, sg1128, and sg0067 from left to right.

[0363] Figure 35 C. Expansion and viability of CD34+ cells derived from peripheral blood of individuals with sickle cell disease after gene editing. Cells were cultured for 6-10 days before electroporation. D0 refers to the day of electroporation. Expansion of total monocytes at days 3, 7, and 10 after editing is shown. N = 4, data show mean values. + SEM. Four independent experiments were performed in duplicate. Bars at each time point are mock, sg1128, and sg0067 from left to right.

[0364] Figure 35 D. Expansion and viability of CD34+ cells derived from peripheral blood of individuals with sickle cell disease after gene editing. Cells were cultured for 6-10 days before electroporation. D0 refers to the day of electroporation. Viability of monocytes at days 3, 7, and 10 after editing is shown. N = 4, data show mean values. + SEM. Four independent experiments were performed in duplicate. Bars at each time point are mock, sg1128, and sg0067 from left to right.

[0365] Figure 36 Editing efficiency of sg1128 and sg0067 in CD34+ cells from sickle cell disease patient samples. The editing pattern of sg0067 is represented by the sum of large deletions (grey) and small insertions / deletions (black). Data show the average of four independent editing experiments performed in duplicate using CD34+ cells from four different sickle cell disease patients (SCD1-4). + SEM.

[0366] Figure 37In vitro multilineage differentiation capacity of HSPCs as measured by colony forming unit assay. BFU-E = primitive forming units, erythroid; CFU-GM = colony forming units, granulocytes, monocytes; CFU-GEMM = colony forming units, granulocytes, erythroid, monocytes, megakaryocytes. Graph shows three independent experiments with CD34+ cells from three different sickle cell disease patients (SCD1-3). Experiments were performed in triplicate. Data represent mean values. + SEM.

[0367] Figure 38 Figure .Fold change of gamma-globin transcripts in erythroid cells from patient samples following CRISPR knockdown of BCL11A or indel creation at the gamma-globin gene cluster. CD34+ cells derived from 3 sickle cell disease patients (SCD1-3) were CRISPR edited and differentiated in vitro into the erythroid lineage as described in the Examples. On day 11 of erythroid differentiation, cells were harvested from culture and qPCR was performed to measure gamma-globin and beta-globin transcripts, normalized to GAPDH. Experiments were performed in duplicate. Data show the average of the pooled donors. + SEM.

[0368] Figure 39 .Counting of HbF+ cells in sickle cell disease patient samples after gene editing. CD34+ cells derived from three sickle cell disease patients (SCD1-3) were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the examples. On days 11, 14, and 21 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody to count HbF+ cells by flow cytometry. The experiment was performed in duplicate. The data show mean ± SEM. Day 11 shows the results from SCD1-3; Day 14 shows the results from SCD-1 and SCD-2; Day 21 shows the results from SCD-2 and SCD-3.

[0369] Figure 40 .Measurement of fetal hemoglobin expression in gene-edited sickle cell disease patient samples by flow cytometry. CD34+ cells derived from three sickle cell disease patients (SCD1-3) were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the examples. On days 11, 14, and 21 of erythroid cell differentiation, cells were stained with anti-HbF-FITC antibody to measure the HbF expression intensity of each cell by flow cytometry. The experiment was performed in duplicate. Data show the mean +SEM. MFI = mean fluorescence intensity. Results from SCD1-3 are shown on day 11; results from SCD-1 and SCD-2 are shown on day 14; and results from SCD-2 and SCD-3 are shown on day 21.

[0370] Figure 41 The number of sickle cells compared to normal cells was counted after CRISPR editing of patient samples. CD34+ cells derived from three sickle cell disease patients (SCD1-3) were CRISPR-edited and differentiated into the erythroid lineage in vitro as described in the previous procedure. On day 21 of erythroid differentiation, cells were placed in a % hypoxia chamber for 4 days, fixed, and then subjected to single-cell imaging flow cytometry. Figure 41 A (left) shows changes in sickle cell numbers in gene-edited patient samples, counted by single-cell imaging. Figure 41 B (right) shows the change in the number of normal cells after gene editing in patient samples counted by single-cell imaging. Three independent experiments were performed, each in duplicate. Forty thousand single-cell images were counted from each patient. The graph shows the average value from the pooled data. + SEM.

[0371] definition

[0372] The term "CRISPR system", "Cas system" or "CRISPR / Cas system" refers to a group of molecules comprising RNA-guided nucleases or other effector molecules and gRNA molecules, which together are necessary and sufficient to guide and achieve modification of nucleic acids at target sequences by RNA-guided nucleases or other effector molecules. In one embodiment, the CRISPR system comprises gRNA and Cas protein (e.g., Cas9 protein). Such systems comprising Cas9 or modified Cas9 molecules are referred to herein as "Cas9 systems" or "CRISPR / Cas9 systems". In one example, the gRNA molecules and Cas molecules can be compounded to form a ribonucleoprotein (RNP) complex.

[0373] The terms "guide RNA," "guide RNA molecule," "gRNA molecule," or "gRNA" are used interchangeably and refer to a set of nucleic acid molecules that facilitate the specific guidance of an RNA-guided nuclease or other effector molecule (typically in complex with a gRNA molecule) to a target sequence. In some embodiments, the guidance is achieved by hybridizing a portion of the gRNA to DNA (e.g., via a gRNA targeting domain) and by binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule (e.g., at least via a gRNA tracr). In an embodiment, the gRNA molecule consists of a single continuous polynucleotide molecule, referred to herein as a "single guide RNA" or "sgRNA," etc. In other embodiments, the gRNA molecule consists of multiple, typically two, polynucleotide molecules that are themselves capable of associating, typically by hybridization, referred to herein as a "dual guide RNA" or "dgRNA," etc. The gRNA molecule is described in more detail below, but typically comprises a targeting domain and a tracr. In an embodiment, the targeting domain and tracr are disposed on a single polynucleotide. In other embodiments, the targeting domain and tracr are disposed on separate polynucleotides.

[0374] The term "targeting domain" (when the term is used in conjunction with a gRNA) is the portion of a gRNA molecule that recognizes, e.g., is complementary to, a target sequence (e.g., a target sequence within a cellular nucleic acid, e.g., within a gene).

[0375] The term "crRNA" (when the term is used in conjunction with a gRNA molecule) is the portion of the gRNA molecule that comprises the targeting domain and the region that interacts with tracr to form the signature stem region.

[0376] The term "target sequence" refers to a nucleic acid sequence that is complementary to the gRNA targeting domain, such as a completely complementary sequence. In an embodiment, the target sequence is arranged on genomic DNA. In one embodiment, the target sequence is adjacent to a protospacer adjacent motif (PAM) sequence (e.g., a PAM sequence recognized by Cas9) recognized by a protein with nuclease or other effector activity (on the same chain of DNA or on the complementary chain of DNA). In an embodiment, the target sequence is a target sequence in a gene or locus that affects globin gene expression, such as beta globulin or fetal hemoglobin (HbF) expression. In an embodiment, the target sequence is a target sequence in a non-deleted HPFH region, such as in an HBG1 and / or HBG2 promoter region.

[0377] The term "flagpole" as used herein in connection with a gRNA molecule refers to the portion of the gRNA where the crRNA and tracr bind or hybridize to each other.

[0378] The term "tracr" as used herein in conjunction with a gRNA molecule refers to the portion of the gRNA that binds to a nuclease or other effector molecule. In an embodiment, tracr comprises a nucleic acid sequence that specifically binds to Cas9. In an embodiment, tracr comprises a nucleic acid sequence that forms part of a marker rod.

[0379] The term "Cas9" or "Cas9 molecule" refers to the enzyme responsible for DNA cleavage from the bacterial type II CRISPR / Cas system. Cas9 also includes wild-type protein and functional and non-functional mutants thereof. In an embodiment, Cas9 is Cas9 from Streptococcus pyogenes.

[0380] The term "complementary" as used in conjunction with nucleic acids refers to the pairing of bases A with T or U and G with C. The term complementary refers to fully complementary nucleic acid molecules, i.e., molecules that are at least 80%, 85%, 90%, 95%, 99% complementary in the form of A with T or U pairs and G with C pairs throughout the reference sequence.

[0381] As used in conjunction with homology-directed repair or homologous recombination, "template nucleic acid" refers to the nucleic acid that is inserted into the modification site by the CRISPR system donor sequence to perform gene repair (insertion) at the cut site.

[0382] "Indels" (as the term is used herein) refer to nucleic acids comprising one or more nucleotide insertions, one or more nucleotide deletions, or a combination of nucleotide insertions and deletions relative to a reference nucleic acid, which are produced after exposure to a composition comprising a gRNA molecule (e.g., a CRISPR system). Indels can be determined by sequencing the nucleic acid after exposure to a composition comprising a gRNA molecule, e.g., by NGS. With respect to the site of an indel, an indel is said to be "at or near" a reference site (e.g., a site complementary to a targeting domain of a gRNA molecule) if the indel comprises at least one insertion or deletion within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of the reference site, or an indel overlaps partially or completely with the reference site (e.g., an indel comprises at least one insertion or deletion that overlaps 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of a site complementary to a targeting domain of a gRNA molecule, e.g., a site complementary to a targeting domain of a gRNA molecule as described herein). In an embodiment, the indel is a large deletion, e.g., comprising 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, greater than about 6 kb, or greater than about 10 kb of nucleic acid. In an embodiment, the 5' end, 3' end, or both the 5' and 3' ends of the large deletion are placed at or near the target sequence of a gRNA molecule described herein. In an embodiment, the large deletion comprises approximately 4.9 kb of DNA (which is placed between the target sequences of a gRNA molecule described herein, for example, in the HBG1 promoter region) and the target sequence of a gRNA molecule described herein, for example, in the HBG2 promoter region.

[0383] "Indel pattern" (when the term is used herein) refers to a set of indels produced after exposure to a composition comprising a gRNA molecule. In one embodiment, the indel pattern consists of the first three indels, based on the frequency of occurrence. In one embodiment, the indel pattern consists of the first five indels, based on the frequency of occurrence. In one embodiment, the indel pattern consists of indels that exist at a frequency of greater than about 1% relative to all sequencing reads. In one embodiment, the indel pattern consists of indels that exist at a frequency of greater than about 5% relative to all sequencing reads. In one embodiment, the indel pattern consists of indels that exist at a frequency of greater than about 10% relative to the total number of indel sequencing reads (i.e., those reads that are not composed of an unmodified reference nucleic acid sequence). In one embodiment, the indel pattern comprises any 3 of the first five most frequently observed indels. The indel pattern can be determined, for example, by the methods described herein, for example, by sequencing cells in a cell population exposed to a gRNA molecule.

[0384] " Off-target insertion / deletion " (when the term is used in this article) refers to an insertion / deletion at or near a site other than the target sequence of the targeting domain of the gRNA molecule. Relative to the sequence of the targeting domain of the gRNA, such sites can include, for example, 1, 2, 3, 4, 5 or more mismatched nucleotides. In an exemplary embodiment, such sites are detected using targeted sequencing of off-target sites predicted via computer simulation or by insertion methods known in the art. With regard to gRNA as described herein, the example of off-target insertion / deletion is an insertion / deletion formed at a sequence outside the HBG1 and / or HBG2 promoter region. In an exemplary embodiment, off-target insertion / deletion is formed in the sequence of a gene, for example, within the coding sequence of a gene.

[0385] The terms "a" and "an" refer to one or more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0386] When referring to a measurable value such as an amount, a time interval, and the like, the term "about" is intended to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for performing the disclosed methods.

[0387] The term "antigen" or "Ag" refers to a molecule that causes an immune response. An immune response can involve the production of antibodies or the activation of specific immunocompetent cells or both. Technicians will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Technicians will understand that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that causes an immune response will therefore encode an "antigen" (when the term is used herein). In addition, those skilled in the art will understand that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is apparent that the present invention includes but is not limited to using partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that cause a desired immune response. In addition, it will be understood that antigens do not need to be encoded by a "gene" at all. It is apparent that antigens can be synthesized or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include but are not limited to tissue samples, cells, or fluids with other biological components.

[0388] The term "autologous" refers to any material derived from the same individual into which it is later introduced.

[0389] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species can be sufficiently genetically different to interact antigenically.

[0390] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0391] "Derived from" as the term is used herein denotes a relationship between a first and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the process or origin of the first molecule derived from the second molecule.

[0392] The term "coding" refers to the intrinsic properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) as a template for synthesizing other polymers and macromolecules with a definite nucleotide sequence (e.g., rRNA, tRNA and mRNA) or definite amino acid sequence in a biological process, and the resulting biological properties. Therefore, if transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and non-coding strand (transcribed as a template for a gene or cDNA) can be referred to as coded protein or other products of the gene or cDNA.

[0393] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that the nucleotide sequence encoding the protein may contain one or more introns in certain versions.

[0394] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition as described herein that is effective to achieve a specific biological result.

[0395] The term "endogenous" refers to any material derived from or produced within an organism, cell, tissue or system.

[0396] The term "exogenous" refers to any material introduced from or generated outside of an organism, cell, tissue, or system.

[0397] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0398] The term "transfer vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic compounds or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as further including non-plasmids and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0399] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0400] The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, such as two nucleic acid molecules (such as two DNA molecules or two RNA molecules) or between two polypeptide molecules. When a subunit position in the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., five positions in a polymer that is ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0401] The term "isolated" means altered or removed from its native state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its native state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment (such as, for example, a host cell).

[0402] The term "operably linked" or "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. DNA sequences that are operably linked can be contiguous to each other and, for example, in the case of linking two protein coding regions, be in the same reading frame.

[0403] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral or infusion techniques.

[0404] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0405] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, such as those also commonly referred to in the art as peptides, oligopeptides, and oligomers; and also to longer chains, such as those commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include naturally occurring peptides, recombinant peptides, or combinations thereof.

[0406] The term "promoter" refers to a DNA sequence recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.

[0407] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also contain enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses the gene product in a tissue-specific manner.

[0408] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0409] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0410] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0411] As used herein in conjunction with messenger RNA (mRNA), a 5' cap (also referred to as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is crucial for being recognized by the ribosome and protected from RNases. Cap addition is coupled to transcription and occurs co-transcriptionally so that each affects the other. Shortly after transcription begins, the 5' end of the synthesized mRNA is bound by a cap synthesis complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis is performed as a multi-step biochemical reaction. The capping portion can be modified to modulate the function of the mRNA, such as its stability or translation efficiency.

[0412] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Typically, in vitro transcribed RNA is produced by an in vitro transcription vector. The in vitro transcription vector comprises a template for producing in vitro transcribed RNA.

[0413] As used herein, "poly (A)" is a series of adenosines attached to mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, the poly A is between 50 and 5000 (SEQ ID NO: 190), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly (A) sequence can be chemically or enzymatically modified to modulate mRNA function, such as localization, stability, or translation efficiency.

[0414] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variant to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly (A) tail is a long sequence of adenine nucleotides (usually hundreds) added to the pre-mRNA by the action of an enzyme (poly (A) polymerase). In higher eukaryotes, the poly (A) tail is added to a transcript containing a specific sequence (polyadenylation signal). The poly (A) tail and the proteins bound thereto help protect the mRNA from being degraded by exonucleases. Polyadenylation is also important for transcription termination, exporting mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after DNA is transcribed into RNA, but may also occur later in the cytoplasm. After transcription has terminated, the mRNA chain is cut by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end at the cleavage site.

[0415] As used herein, "transient" refers to expression of a non-integrated transgene for hours, days, or weeks, where the period of expression is less than the period of expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0416] As used herein, the terms "treat, treatment and treating" refer to reducing or alleviating the progression, severity and / or duration of a condition, such as a hemoglobinopathy, or alleviating one or more symptoms (preferably, one or more discernible symptoms) of a condition, such as a hemoglobinopathy, caused by the administration of one or more therapies (e.g., one or more therapeutic agents, such as the gRNA molecules, CRISPR systems or modified cells of the present invention). In specific embodiments, the terms "treat, treatment and treating" refer to improving at least one measurable physical parameter of a hemoglobinopathy condition that is not discernible to the patient. In other embodiments, the terms "treat, treatment and treating" refer to inhibiting the progression of a condition physically, for example, by stabilizing a discernible symptom, physiologically, for example, by stabilizing a physical parameter, or both. In other embodiments, the terms "treat, treatment and treating" refer to reducing or stabilizing the symptoms of a hemoglobinopathy, such as sickle cell disease or beta-thalassemia.

[0417] The term "signal transduction pathway" refers to the biochemical relationships between various signal transduction molecules that play a role in transmitting a signal from one part of a cell to another. The phrase "cell surface receptor" includes molecules and molecular complexes that are capable of receiving a signal and transmitting the signal across the cell membrane.

[0418] The term "subject" is intended to include living organisms (eg, mammals, humans) in which an immune response can be elicited.

[0419] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a substantially purified cell population refers to a homogenous cell population. In other cases, the term refers only to cells that have been separated from cells with which they are naturally associated in their naturally occurring state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0420] As used herein, the term "therapeutic" means treatment. A therapeutic effect is achieved by reducing, inhibiting, alleviating or eradicating a disease state.

[0421] As used herein, the term "prevention" means prophylactic or protective treatment against a disease or disease state.

[0422] The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acids and / or proteins into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids and / or proteins. Cells include primary host cells and their progeny.

[0423] The term "specifically binds" refers to a molecule that recognizes and binds to a binding partner (eg, a protein or nucleic acid) present in a sample, but does not substantially recognize or bind other molecules in the sample.

[0424] The term "bioequivalent" refers to the amount of an agent other than a reference compound required to produce an effect comparable to the effect produced by a reference dose or reference amount of a reference compound.

[0425] As used herein, "refractory" refers to a disease, such as a hemoglobinopathy, that is unresponsive to treatment. In embodiments, a refractory hemoglobinopathy may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory hemoglobinopathy may become resistant during treatment. Refractory hemoglobinopathy is also known as a resistant hemoglobinopathy.

[0426] As used herein, "relapse" refers to the return of a disease (eg, a hemoglobinopathy) or signs and symptoms of a disease (eg, a hemoglobinopathy after a period of improvement, eg, a hemoglobinopathy after previous treatment with a therapy (eg, a hemoglobinopathy therapy)).

[0427] Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the invention. Thus, descriptions of ranges should be considered to include all possible subranges as well as individual numerical values ​​within the range as specifically disclosed. For example, a description of a range such as from 1 to 6 should be considered to include subranges as specifically disclosed as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numerical values ​​within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95%-99% identity includes 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96%-99%, 96%-98%, 96%-97%, 97%-99%, 97%-98%, and 98%-99% identity. This applies regardless of the width of the range.

[0428] The term "BCL11a" refers to B-cell lymphoma / leukemia 11A (an RNA polymerase II core promoter proximal region sequence-specific DNA binding protein) and the gene encoding the protein, as well as all introns and exons. This gene encodes a C2H2-type zinc finger protein. BCL11A has been found to play a role in inhibiting fetal hemoglobin production. BCL11a is also known as B-cell CLL / lymphoma 11A (zinc finger protein), CTIP1, EVI9, ecotropic viral integration site 9 protein homolog, COUP-TF-interacting protein 1, zinc finger protein 856, KIAA1809, BCL-11A, ZNF856, EVI-9, and B-cell CLL / lymphoma 11A. This term encompasses all isoforms and splice variants of BCL11a. The human gene encoding BCL11a is mapped to chromosome position 2p16.1 (by Ensembl). Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. The human BCL11a genomic sequence can be found in GenBank at NC_000002.12. The BCL11a gene refers to this genomic location, including all introns and exons. There are multiple known isoforms of BCL11a.

[0429] The mRNA sequence encoding isoform 1 of human BCL11a can be found at NM_022893.

[0430] The peptide sequence of isoform 1 of human BCL11a is:

[0431]

[0432] SEQ ID NO: 245 (identifiers Q9H165-1; and NM_022893.3; and accession number ADL14508.1). Sequences of other BCL11a protein isoforms are provided at:

[0433] Isotype 2: Q9H165-2

[0434] Isotype 3: Q9H165-3

[0435] Isotype 4: Q9H165-4

[0436] Isotype 5: Q9H165-5

[0437] Isotype 6: Q9H165-6

[0438] As used herein, human BCL11a protein also encompasses proteins having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to BCL11a isoforms 1-6 over their full length, wherein such proteins still possess at least one function of BCL11a.

[0439] As used herein, the term "globin locus" refers to the region of human chromosome 11 that contains the embryonic (ε), fetal (G(γ) and A(γ)) genes, the adult globin genes (γ and β), the locus control region, and the DNase I hypersensitivity site.

[0440] The term "complementary" as used in conjunction with nucleic acids refers to the pairing of bases A with T or U and G with C. The term complementary refers to fully complementary nucleic acid molecules, i.e., molecules that are at least 80%, 85%, 90%, 95%, 99% complementary in the form of A with T or U pairs and G with C pairs throughout the reference sequence.

[0441] The term "non-deletional HPFH" refers to a mutation that does not contain an insertion or deletion of one or more nucleotides, which results in an inherited persistence of fetal hemoglobin and is characterized by increased fetal hemoglobin in adult erythrocytes. In an exemplary embodiment, non-deletional HPFH is a mutation described in Nathan and Oski, Hematology and Oncology of Infancy and Childhood, 8th Ed. [Orkin SH, Fisher DE, Look T, Lux SE, Ginsburg D, Nathan DG, eds., Elsevier Saunders (incorporated herein by reference in its entirety)], 2015. Non-deletion HPFH mutations are described, for example, in Table 21-5. The term "non-deletion HPFH region" refers to a genomic locus containing or proximal to a non-deletion HPFH region. In an exemplary embodiment, the non-deletion HPFH region is the nucleic acid sequence of the HBG1 promoter region (Chr 11:5,249,833 to Chr 11:5,250,237, hg38; -strand), the nucleic acid sequence of the HBG2 promoter region (Chr 11:5,254,738 to Chr 11:5,255,164, hg38; -strand), or a combination thereof. In an exemplary embodiment, the non-deletion HPFH region includes Nathan and Oski's Hematology and Oncology of Infancy and Childhood, 8 th One or more of the non-deletional HPFH described in Orkin's Infant and Childhood Hematology and Oncology, 8th ed., 2015, Orkin SH, Fisher DE, Look T, Lux SE, Ginsburg D, Nathan DG, eds., Elsevier Saunders (e.g., described in Table 21-5). In an exemplary embodiment, the non-deleted HPFH region is at a nucleic acid sequence at chr11:5,250,094-5,250,237,-strand, hg38; or at a nucleic acid sequence at chr11:5,255,022-5,255,164,-strand, hg38; or at a nucleic acid sequence at chr11:5,249,833-5,249,927,-strand, hg38; or at a nucleic acid sequence at chr11:5,254,738-5,254,851,-strand, hg38; or at a nucleic acid sequence at chr11:5,250,139-5,250,237,-strand, hg38; or a combination thereof.

[0442] "BCL11a enhancer" (when the term is used herein) refers to a nucleic acid sequence that affects, for example, the expression or function of BCL11a. See, for example, Bauer et al., Science, Vol. 342, 2013, pp. 253-257. A BCL11a enhancer may, for example, be effective only in certain cell types (e.g., erythroid lineage cells). An example of a BCL11a enhancer is a nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene (e.g., located at or corresponding to positions +55: Chr2: 60497676-60498941; +58: Chr2: 60494251-60495546; +62: Chr2: 60490409-60491734 as recorded in hg38). In one embodiment, the BCL11a enhancer is the +62 region of the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene. In one embodiment, the BCL11a enhancer is the +58 region of the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene. In one embodiment, the BCL11a enhancer is the +55 region of the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene.

[0443] The terms "hematopoietic stem and progenitor cells" or "HSPCs" are used interchangeably and refer to a cell population comprising both hematopoietic stem cells ("HSCs") and hematopoietic progenitor cells ("HPCs"). Such cells are characterized as, for example, CD34+. In exemplary embodiments, HSPCs are isolated from bone marrow. In other exemplary embodiments, HSPCs are isolated from peripheral blood. In other exemplary embodiments, HSPCs are isolated from umbilical cord blood. In one embodiment, HSPCs are characterized as CD34+ / CD38- / CD90+ / CD45RA-. In an embodiment, HSPCs are characterized as CD34+ / CD90+ / CD49f+ cells. In an embodiment, HSPCs are characterized as CD34+ cells. In an embodiment, HSPCs are characterized as CD34+ / CD90+ cells. In an embodiment, HSPCs are characterized as CD34+ / CD90+ cells. In an embodiment, HSPCs are characterized as CD34+ / CD90+ cells.

[0444] As used herein, a "stem cell expander" refers to a compound that causes cells (e.g., HSPCs, HSCs, and / or HPCs) to proliferate (e.g., increase in number) at a faster rate than the same cell type in the absence of the agent. In one exemplary aspect, the stem cell expander is an inhibitor of the aryl hydrocarbon receptor pathway. Additional examples of stem cell expanders are provided below. In embodiments, proliferation, e.g., increase in number, is accomplished ex vivo.

[0445] "Engraftment" or "engraft" refers to the incorporation of cells or tissues (e.g., HSPC populations) into a recipient (e.g., a mammal or human subject). In one example, engraftment includes growth, expansion, and / or differentiation of the implanted cells in the recipient. In one example, engraftment of HSPCs includes differentiation and growth of the HSPCs into erythroid cells in the recipient.

[0446] The term "hematopoietic progenitor cell" (HPC) as used herein refers to a primitive hematopoietic cell with limited self-renewal capacity and the potential for multi-lineage differentiation (e.g., myeloid, lymphoid), single-lineage differentiation (e.g., myeloid or lymphoid), or cell type-restricted differentiation (e.g., erythroid progenitor) depending on placement within the hematopoietic hierarchy (Doulatov et al., Cell Stem Cell 2012).

[0447] As used herein, "hematopoietic stem cell" (HSC) refers to immature blood cells that have the ability to self-renew and differentiate into more mature blood cells, including granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, thrombopoietic megakaryocytes, platelets) and monocytes (e.g., monocytes, macrophages). Throughout the specification, HSCs are interchangeably described as stem cells. It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. It is believed that CD34+ cells include a subpopulation of cells having the stem cell properties defined above. As is well known in the art, HSCs are multipotent cells that can produce primitive progenitor cells (e.g., multipotent progenitor cells) and / or progenitor cells (e.g., lymphoid progenitor cells) directed to a specific hematopoietic lineage. The stem cells directed to a specific hematopoietic lineage can belong to a T cell lineage, a B cell lineage, a dendritic cell lineage, a Langerhans cell lineage and / or a lymphoid tissue-specific macrophage lineage. In addition, HSC also refers to long-term HSC (LT-HSC) and short-term HSC (ST-HSC). ST-HSC is more active and more proliferative than LT-HSC. However, LT-HSC has unlimited self-renewal (that is, they survive throughout adulthood), while ST-HSC has limited self-renewal (that is, they only survive a limited period of time). Any of these HSCs can be used in any method as described herein. Optionally, ST-HSC is useful because they have high proliferative properties, and therefore increase the quantity of HSC and its offspring rapidly. Hematopoietic stem cells are optionally obtained from blood products. Blood products include products obtained from body organs of cells in vivo or containing hematopoietic origin. Such sources include unfractionated bone marrow, umbilical cord, peripheral blood (for example mobilized peripheral blood, for example with mobilizing agents such as G-CSF or (AMD3100), or G-CSF and (AMD3100) combination mobilized peripheral blood), liver, thymus, lymph nodes, and spleen. All of the above crude or unfractionated blood products can be enriched for cells with hematopoietic stem cell characteristics using methods known to those skilled in the art. In one embodiment, HSCs are characterized as CD34+ / CD38- / CD90+ / CD45RA-. In an embodiment, HSCs are characterized as CD34+ / CD90+ / CD49f+ cells. In an embodiment, HSCs are characterized as CD34+ cells. In an embodiment, HSCs are characterized as CD34+ / CD90+ cells. In an embodiment, HSCs are characterized as CD34+ / CD90+ cells. In an embodiment, HSCs are characterized as CD34+ / CD90+ cells.

[0448] "Expansion" or "expand" in the context of cells refers to an increase in the number of a specific cell type or types from an initial cell population of cells (which may be the same or different). The initial cells used for expansion may be different from the cells produced from the expansion.

[0449] A "cell population" refers to eukaryotic mammalian (preferably human) cells isolated from a biological source (eg, a blood product or tissue) and derived from more than one cell type.

[0450] When used in the context of a cell population, "enriched" refers to a cell population that is selected based on the presence of one or more markers (eg, CD34+).

[0451] The term "CD34+ cells" refers to cells that express the CD34 marker on their surface. CD34+ cells can be detected and counted using, for example, flow cytometry and fluorescently labeled anti-CD34 antibodies.

[0452] By "enriched in CD34+ cells" is meant a cell population that has been selected based on the presence of the CD34 marker. Thus, the percentage of CD34+ cells in the cell population after the selection process is higher than the percentage of CD34+ cells in the initial cell population prior to the selection step based on the CD34 marker. For example, CD34+ cells can represent at least 50%, 60%, 70%, 80%, or at least 90% of the cells in the cell population enriched for CD34+ cells.

[0453] The terms "F cells" and "F-cells" refer to cells that contain and / or produce (e.g., express) fetal hemoglobin, typically red blood cells (e.g., erythrocytes). For example, F-cells are cells that contain or produce detectable levels of fetal hemoglobin. For example, F-cells are cells that contain or produce at least 5 picograms of fetal hemoglobin. In another example, F-cells are cells that contain or produce at least 6 picograms of fetal hemoglobin. In another example, F-cells are cells that contain or produce at least 7 picograms of fetal hemoglobin. In another example, F-cells are cells that contain or produce at least 8 picograms of fetal hemoglobin. In another example, F-cells are cells that contain or produce at least 9 picograms of fetal hemoglobin. In another example, F-cells are cells that contain or produce at least 10 picograms of fetal hemoglobin. The level of fetal hemoglobin can be measured using assays described herein or by other methods known in the art, such as flow cytometry, high performance liquid chromatography, mass spectrometry, or enzyme-linked immunosorbent assay using anti-fetal hemoglobin detection reagents.

[0454] Unless otherwise stated, all genomic or chromosomal coordinates are according to hg38. DETAILED DESCRIPTION

[0455] The gRNA molecules, compositions and methods described herein relate to genome editing in eukaryotic cells using the CRISPR / Cas9 system. Specifically, the gRNA molecules, compositions and methods described herein relate to the regulation of globin levels and can be used, for example, to regulate the expression and production of globin genes and proteins. These gRNA molecules, compositions and methods can be used to treat hemoglobinopathies.

[0456] I. gRNA molecules

[0457] The gRNA molecule can have many domains, as described more fully below, however, the gRNA molecule generally comprises at least one crRNA domain (comprising a targeting domain) and tracr. The gRNA molecule of the present invention used as a component of the CRISPR system can be used to modify (e.g., a modified sequence) DNA at or near the target site. Such modifications include deletions and or insertions that result in, for example, reduced or eliminated expression of a functional product of a gene comprising a target site. These uses and other uses are described more fully below.

[0458] In one embodiment, a single molecule or sgRNA preferably comprises, from 5' to 3': a crRNA (containing a targeting domain complementary to the target sequence and a region forming part of a marker stem (i.e., a crRNA marker stem region)); a loop; and a tracr (containing a domain complementary to the crRNA marker stem region and a domain that additionally binds a nuclease or other effector molecule (e.g., a Cas molecule, e.g., a Cas9 molecule)); and can take the following form (from 5' to 3'):

[0459] [Targeting domain]-[crRNA signature stem region]-[optional first signature stem extension]-[loop]-[optional first tracr extension]-[tracr signature stem region]-[tracr nuclease binding domain].

[0460] In an embodiment, the tracr nuclease binding domain binds a Cas protein, e.g., a Cas9 protein.

[0461] In one embodiment, the double molecule or dgRNA comprises two polynucleotides; the first, preferably from 5' to 3': crRNA (which contains a targeting domain complementary to the target sequence and a region forming part of the marker stem); and the second, preferably from 5' to 3': tracr (which contains a domain complementary to the crRNA marker stem region and a domain that additionally binds a nuclease or other effector molecule (e.g., a Cas molecule, such as a Cas9 molecule)); and can take the following forms (from 5' to 3'):

[0462] Polynucleotide 1 (crRNA): [targeting domain]-[crRNA marker region]-[optional first marker extension]-[optional second marker extension]

[0463] Polynucleotide 2 (tracr): [optional first tracr extension]-[tracr marker region]-[tracr nuclease binding domain]

[0464] In an embodiment, the tracr nuclease binding domain binds a Cas protein, e.g., a Cas9 protein.

[0465] In some aspects, the targeting domain comprises or consists of a targeting domain sequence described herein, e.g., a targeting domain described in Table 1, or a targeting domain comprising or consisting of 17, 18, 19, or 20 (preferably 20) consecutive nucleotides of a targeting domain sequence described in Table 1.

[0466] In some aspects, the marker stem (eg, crRNA marker stem region) comprises, from 5' to 3': GUUUUAGAGCUA (SEQ ID NO: 182).

[0467] In some aspects, the marker stem (eg, crRNA marker stem region) comprises, from 5' to 3': GUUUAAGAGCUA (SEQ ID NO: 183).

[0468] In some aspects, the loop comprises from 5' to 3': GAAA (SEQ ID NO: 186).

[0469] In some aspects, tracr comprises from 5' to 3': UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 187), and is preferably used in a gRNA molecule comprising SEQ ID NO 182.

[0470] In some aspects, tracr comprises from 5' to 3': UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 188), and is preferably used in a gRNA molecule comprising SEQ ID NO 183.

[0471] In some aspects, gRNA can also include additional U nucleic acids at the 3' end. For example, gRNA can include additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U nucleic acids (SEQ ID NO: 249) at the 3' end. In one embodiment, gRNA includes additional 4 U nucleic acids at the 3' end. In the case of dgRNA, one or more polynucleotides of dgRNA (for example, a polynucleotide comprising a targeting domain and a polynucleotide comprising tracr) can include additional U nucleic acids at the 3' end. For example, in the case of dgRNA, one or more polynucleotides of dgRNA (for example, a polynucleotide comprising a targeting domain and a polynucleotide comprising tracr) can include additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U nucleic acids (SEQ ID NO: 249) at the 3' end. In one embodiment, in the case of dgRNA, one or more polynucleotides of dgRNA (for example, a polynucleotide comprising a targeting domain and a polynucleotide comprising tracr) include additional 4 U nucleic acids at the 3' end. In one embodiment of the dgRNA, only the polynucleotide comprising tracr comprises additional one or more U nucleic acids, such as 4 U nucleic acids. In one embodiment of the dgRNA, only the polynucleotide comprising the targeting domain comprises additional one or more U nucleic acids. In one embodiment of the dgRNA, both the polynucleotide comprising the targeting domain and the polynucleotide comprising tracr comprise additional U nucleic acids, such as 4 U nucleic acids.

[0472] In some aspects, gRNA can also include additional A nucleic acid at the 3' end. For example, gRNA can include additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 A nucleic acid (SEQ ID NO: 250) at the 3' end. In one embodiment, gRNA includes additional 4 A nucleic acid at the 3' end. In the case of dgRNA, one or more polynucleotides of dgRNA (for example, a polynucleotide comprising a targeting domain and a polynucleotide comprising tracr) can include additional A nucleic acid at the 3' end. For example, in the case of dgRNA, one or more polynucleotides of dgRNA (for example, a polynucleotide comprising a targeting domain and a polynucleotide comprising tracr) can include additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 A nucleic acid (SEQ ID NO: 250) at the 3' end. In one embodiment, in the case of dgRNA, one or more polynucleotides of dgRNA (for example, a polynucleotide comprising a targeting domain and a polynucleotide comprising tracr) include additional 4 A nucleic acid at the 3' end. In one embodiment of the dgRNA, only the polynucleotide comprising tracr comprises additional one or more A nucleic acids, such as 4 A nucleic acids. In one embodiment of the dgRNA, only the polynucleotide comprising the targeting domain comprises additional one or more A nucleic acids. In one embodiment of the dgRNA, both the polynucleotide comprising the targeting domain and the polynucleotide comprising tracr comprise additional U nucleic acids, such as 4 A nucleic acids.

[0473] In an embodiment, one or more polynucleotides of the gRNA molecule may comprise a cap at the 5' end.

[0474] In one embodiment, a single molecule or sgRNA preferably comprises, from 5' to 3': a crRNA (containing a targeting domain complementary to a target sequence); a crRNA marker region; a first marker extension; a loop; a first tracr extension (containing a domain complementary to at least a portion of the first marker extension); and a tracr (containing a domain complementary to the crRNA marker region and a domain that additionally binds to a Cas9 molecule). In some aspects, the targeting domain comprises a targeting domain sequence described herein, e.g., a targeting domain described in Table 1, or a targeting domain comprising or consisting of 17, 18, 19, or 20 (preferably 20) consecutive nucleotides of a targeting domain sequence described in Table 1 (e.g., 3' 17, 18, 19, or 20 (preferably 20) consecutive nucleotides of a targeting domain sequence described in Table 1).

[0475] In aspects comprising a first marker pole extension and / or a first tracr extension, the marker pole, loop, and tracr sequences can be as described above. Generally, any first marker pole extension and first tracr extension can be used, provided they are complementary. In embodiments, the first marker pole extension and the first tracr extension consist of 3, 4, 5, 6, 7, 8, 9, 10 or more complementary nucleotides.

[0476] In some aspects, the first marker pole extension comprises, from 5' to 3': UGCUG (SEQ ID NO: 184). In some aspects, the first marker pole extension consists of SEQ ID NO: 184.

[0477] In some aspects, the first tracr extension comprises, from 5' to 3': CAGCA (SEQ ID NO: 189). In some aspects, the first tracr extension consists of SEQ ID NO: 189.

[0478] In one embodiment, dgRNA comprises two nucleic acid molecules. In some aspects, dgRNA comprises a first nucleic acid, preferably from 5' to 3' containing: a targeting domain complementary to the target sequence; a crRNA marker rod region; optionally a first marker rod extension; and optionally a second marker rod extension; and a second nucleic acid (which may be referred to herein as tracr, and comprising at least one domain that binds to a Cas molecule (e.g., a Cas9 molecule)), preferably from 5' to 3' comprising: optionally a first tracr extension; and tracr (which contains a domain complementary to the crRNA marker rod region, and additionally binds to a domain of a Cas (e.g., Cas9) molecule). The second nucleic acid may additionally comprise additional U nucleic acids at the 3' end (e.g., 3' of tracr). For example, tracr may comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U nucleic acids (SEQ ID NO: 249) at the 3' end (e.g., 3' of tracr). The second nucleic acid may additionally or alternatively comprise additional A nucleic acids at the 3' end (e.g., 3' of tracr). For example, a tracr can comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 A nucleic acids (SEQ ID NO: 250) at the 3' end (e.g., 3' of tracr). In some aspects, the targeting domain comprises a targeting domain sequence described herein, e.g., a targeting domain described in Table 1, or a targeting domain comprising or consisting of 17, 18, 19, or 20 (preferably 20) consecutive nucleotides of a targeting domain sequence described in Table 1.

[0479] In aspects involving dgRNA, the crRNA flag stem region, optional first flag stem extension, optional first tracr extension, and tracr sequence can be as described above.

[0480] In some aspects, the optional second marker pole extension comprises, from 5' to 3': UUUUG (SEQ ID NO: 185).

[0481] In embodiments, the 3' 1, 2, 3, 4, or 5 nucleotides, the 5' 1, 2, 3, 4, or 5 nucleotides, or both the 3' and 5' 1, 2, 3, 4, or 5 nucleotides of the gRNA molecule (and in the case of a dgRNA molecule, the polynucleotide comprising a targeting domain and / or the polynucleotide comprising a tracr) are modified nucleic acids, as described more fully in Section XIII, below.

[0482] These domains are briefly discussed below:

[0483] 1) Targeting domain:

[0484] Guidance on the selection of targeting domains can be found, for example, in Fu Y et al. NAT BIOTECHNOL 2014 (doi: 10.1038 / nbt.2808) and Sternberg SH et al. NATURE 2014 (doi: 10.1038 / nature13011).

[0485] The targeting domain comprises a target sequence complementary to the target nucleic acid, such as at least 80%, 85%, 90%, 95% or 99% complementary, such as a completely complementary nucleotide sequence. The targeting domain is part of an RNA molecule, so it will include base uracil (U), and any DNA encoding the gRNA molecule will include base thymine (T). Although it is not desired to be bound by theory, it is believed that the complementarity of the targeting domain and the target sequence contributes to the specificity of the interaction of gRNA molecule / Cas9 molecule complex with the target nucleic acid. It should be understood that in the targeting domain and target sequence pairs, the uracil bases in the targeting domain will be paired with the adenine bases in the target sequence.

[0486] In one embodiment, the length of the targeting domain is 5 to 50, such as 10 to 40, such as 10 to 30, such as 15 to 30, such as 15 to 25 nucleotides. In one embodiment, the length of the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In one embodiment, the length of the targeting domain is 16 nucleotides. In one embodiment, the length of the targeting domain is 17 nucleotides. In one embodiment, the length of the targeting domain is 18 nucleotides. In one embodiment, the length of the targeting domain is 19 nucleotides. In one embodiment, the length of the targeting domain is 20 nucleotides. In an embodiment, the above-mentioned 16, 17, 18, 19 or 20 nucleotides comprise 5'-16, 17, 18, 19 or 20 nucleotides from the targeting domain described in Table 1. In an embodiment, the aforementioned 16, 17, 18, 19 or 20 nucleotides comprise 3'-16, 17, 18, 19, or 20 nucleotides from a targeting domain described in Table 1.

[0487] Without being bound by theory, it is believed that the 8, 9, 10, 11 or 12 nucleic acids of the targeting domain disposed at the 3' end of the targeting domain are important for targeting the target sequence and can therefore be referred to as the "core" region of the targeting domain. In one embodiment, the core domain is fully complementary to the target sequence.

[0488] In some aspects, the target nucleic acid chain complementary to the targeting domain is referred to as target sequence in this article.In some aspects, the target sequence is arranged on chromosome, for example, is the target in gene.In some aspects, the target sequence is arranged in the exon of gene.In some aspects, the target sequence is arranged in the intron of gene.In some aspects, the target sequence comprises or approaches the binding site of the regulatory element of (for example, in 10,20,30,40,50,100,200,300,400,500 or 1000 nucleic acids) target gene, for example, promoter or transcription factor binding site.Some or all of the nucleotides in the domain can have modification, for example, the modification found in this paper part XIII.

[0489] 2) crRNA marker region:

[0490] The marker rod contains portions from both crRNA and tracr. The crRNA marker rod region is complementary to a portion of tracr, and in one embodiment, has sufficient complementarity with a portion of tracr to form a double-stranded region under at least some physiological conditions (e.g., normal physiological conditions). In one embodiment, the length of the crRNA marker rod region is 5 to 30 nucleotides. In one embodiment, the length of the crRNA marker rod region is 5 to 25 nucleotides. The crRNA marker rod region may have homology with a naturally occurring portion of a repetitive sequence from a bacterial CRISPR array, or may be derived from a naturally occurring portion of a repetitive sequence from a bacterial CRISPR array. In one embodiment, it has at least 50% homology with a crRNA marker rod region disclosed herein (e.g., a Streptococcus pyogenes or Streptococcus thermophilus crRNA marker rod region).

[0491] In one embodiment, the marker stem (e.g., crRNA marker stem region) comprises SEQ ID NO: 182. In one embodiment, the marker stem (e.g., crRNA marker stem region) comprises a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homologous to SEQ ID NO: 182. In one embodiment, the marker stem (e.g., crRNA marker stem region) comprises at least 5, 6, 7, 8, 9, 10, or 11 nucleotides of SEQ ID NO: 182. In one embodiment, the marker stem (e.g., crRNA marker stem region) comprises SEQ ID NO: 183. In one embodiment, the marker stem comprises a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homologous to SEQ ID NO: 183. In one embodiment, the marker stem (e.g., crRNA marker stem region) comprises at least 5, 6, 7, 8, 9, 10, or 11 nucleotides of SEQ ID NO: 183.

[0492] Some or all of the nucleotides of a domain may have modifications, such as those described in Section XIII herein.

[0493] 3) First signpost extension

[0494] When a tracr comprising a first tracr extension is used, the crRNA may comprise a first marker stem extension. Generally, any first marker stem extension and first tracr extension may be used, provided they are complementary. In an embodiment, the first marker stem extension and the first tracr extension consist of 3, 4, 5, 6, 7, 8, 9, 10 or more complementary nucleotides.

[0495] The first marker pole extension can comprise nucleotides that are complementary to the nucleotides of the first tracr extension, e.g., 80%, 85%, 90%, 95%, or 99% complementary, e.g., fully complementary. In some aspects, the first marker pole extension nucleotides that hybridize to the complementary nucleotides of the first tracr extension are contiguous. In some aspects, the first marker pole extension nucleotides that hybridize to the complementary nucleotides of the first tracr extension are discontinuous, e.g., comprise two or more hybridization regions separated by nucleotides that do not base pair with the nucleotides of the first tracr extension. In some aspects, the first marker pole extension comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some aspects, the first marker pole extension comprises, from 5' to 3', UGCUG (SEQ ID NO: 184). In some aspects, the first marker pole extension consists of SEQ ID NO: 184. In some aspects, the first marker pole extension comprises a nucleic acid that is at least 80%, 85%, 90%, 95%, or 99% homologous to SEQ ID NO:184.

[0496] Some or all of the nucleotides of the first tracr extension can have modifications, such as those found in Section XIII herein.

[0497] 3) Ring

[0498] The ring is used to connect the crRNA flag rod region (or optionally the first flag rod extension, when present) to the tracr of the sgRNA (or optionally the first tracr extension, when present). The ring can covalently or non-covalently connect the crRNA flag rod region and tracr. In one embodiment, the connection is covalent. In one embodiment, the ring covalently couples the crRNA flag rod region and tracr. In one embodiment, the ring covalently couples the first flag rod extension and the first tracr extension. In one embodiment, the ring is or comprises a covalent bond inserted between the crRNA flag rod region and the domain of tracr that hybridizes with the crRNA flag rod region. Typically, the ring comprises one or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0499] In a dgRNA molecule, the two molecules can associate by virtue of hybridization between at least a portion of the crRNA (e.g., the crRNA marker stem region) and at least a portion of tracr (e.g., a domain of tracr that is complementary to the crRNA marker stem region).

[0500] A variety of loops are suitable for use in sgRNA. The loop can be composed of a covalent bond, or as short as one or several nucleotides, for example, 1, 2, 3, 4 or 5 nucleotides in length. In one embodiment, the length of the loop is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides. In one embodiment, the length of the loop is 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 has homology with a naturally occurring sequence, or is derived from a naturally occurring sequence. In one embodiment, the loop has at least 50% homology with a loop disclosed herein. In one embodiment, the loop comprises SEQ ID NO: 186.

[0501] Some or all of the nucleotides of a domain may have modifications, such as those described in Section XIII herein.

[0502] 4) Second sign pole extension

[0503] In one embodiment, the dgRNA may comprise an additional sequence (3' at the crRNA marker region), or (when present) a first marker extension, referred to herein as a second marker extension. In one embodiment, the length of the second marker extension is 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides. In one embodiment, the length of the second marker extension is 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. In one embodiment, the second marker extension comprises SEQ ID NO: 185.

[0504] 5) Tracr:

[0505] Tracr is a nucleic acid sequence required for a nuclease (such as Cas9) to bind. Without being bound by theory, it is believed that 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 comprises a sequence from or derived from Streptococcus pyogenes tracr. In some aspects, tracr has a portion that hybridizes with a marker rod portion of crRNA, for example, having enough complementarity with the crRNA marker rod region to form a double-stranded region (sometimes referred to herein as a tracr marker rod region or a tracr domain complementary to the crRNA marker rod region) under at least some physiological conditions. In an embodiment, the domain of tracr that hybridizes with the crRNA marker rod region comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides that hybridize with the complementary nucleotides of the crRNA marker rod region. In some aspects, the tracr nucleotides that hybridize with the complementary nucleotides of the crRNA marker rod region are continuous. In some aspects, the tracr nucleotides that hybridize to the complementary nucleotides of the crRNA marker rod region are discontinuous, for example, comprising two or more hybridization regions separated by nucleotides that are not base-paired with the nucleotides of the crRNA marker rod region. In some aspects, the portion of tracr that hybridizes to the crRNA marker rod region comprises from 5' to 3': UAGCAAGUUAAAA (SEQ ID NO: 191). In some aspects, the portion of tracr that hybridizes to the crRNA marker rod region comprises from 5' to 3': UAGCAAGUUUAAA (SEQ ID NO: 192). In an embodiment, the sequence that hybridizes to the crRNA marker rod region is arranged on tracr, 5'- to the sequence of the tracr that additionally binds to the nuclease (e.g., Cas molecule, e.g., Cas9 molecule).

[0506] Tracr further comprises a domain that binds to a nuclease (e.g., a Cas molecule, such as a Cas9 molecule). Without being bound by theory, it is believed that Cas9 from different species bind to different tracr sequences. In some aspects, tracr comprises a sequence that binds to a Streptococcus pyogenes Cas9 molecule. In some aspects, tracr comprises a sequence that binds to a Cas9 molecule disclosed herein. In some aspects, the domain that binds to the Cas9 molecule comprises from 5' to 3': UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 193). In some aspects, the domain that binds to the Cas9 molecule comprises from 5' to 3': UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 194).

[0507] In some embodiments, the tracr comprises SEQ ID NO: 187. In some embodiments, the tracr comprises SEQ ID NO: 188.

[0508] Some or all of the nucleotides of tracr can have modifications, such as those found in Section XIII herein. In an embodiment, gRNA (e.g., sgRNA or dgRNA tracr and / or crRNA), such as any of the above-mentioned gRNA or gRNA components, comprises reverse abasic residues at the 5' end, 3' end, or 5' and 3' ends of gRNA. In an embodiment, gRNA (e.g., sgRNA or dgRNA tracr and / or crRNA), such as any of the above-mentioned gRNA or gRNA components, comprises one or more phosphorothioate bonds between the 5' end residues of a polynucleotide, such as 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 an embodiment, gRNA or gRNA components can alternatively or additionally be included in one or more phosphorothioate bonds between the 3' end residues of a polynucleotide, such as between the first two 3' residues, between each of the first three 3' residues, between each of the first four 3' residues, or between each of the first five 3' residues. In one embodiment, gRNA (e.g., sgRNA or dgRNA tracr and / or crRNA), such as any of the above gRNA or gRNA components, comprises a phosphorothioate bond between each of the first four 5' residues (e.g., three phosphorothioate bonds included at the 5' end, e.g., consisting of thereof) and a phosphorothioate bond between each of the first four 3' residues (e.g., three phosphorothioate bonds included at the 3' end, e.g., consisting of thereof). In one embodiment, any of the above phosphorothioate modifications are combined with the reverse abasic residues at the 5' end, 3' end, or 5' and 3' ends of the polynucleotide. In such embodiments, the reverse abasic nucleotides can be connected to the 5' and / or 3' nucleotides by a phosphate bond or a phosphorothioate bond. In an embodiment, gRNA (e.g., sgRNA or dgRNA tracr and / or crRNA), such as any of the above gRNA or gRNA components, comprises one or more nucleotides containing a 2'O-methyl modification. In an embodiment, each of the first 1, 2, 3 or more of the 5' residues comprises a 2'O-methyl modification. In an embodiment, each of the first 1, 2, 3 or more of the 3' residues comprises a 2'O-methyl modification. In an embodiment, the 4th, 3rd and 2nd 3' residues relative to the end comprise a 2'O-methyl modification. In an embodiment, each of the first 1, 2, 3 or more of the 5' residues comprises a 2'O-methyl modification, and each of the first 1, 2, 3 or more of the 3' residues comprises a 2'O-methyl modification. In an embodiment, each of the first 3 of the 5' residues comprises a 2'O-methyl modification, and each of the first 3 of the 3' residues comprises a 2'O-methyl modification.In an embodiment, each of the first three of the 5' residues comprises a 2'O-methyl modification, and the 4th relative to the end, the 3rd relative to the end, and the 2nd 3' residue relative to the end comprise a 2'O-methyl modification. In an embodiment, any 2'O-methyl modification, such as described above, can be combined with one or more phosphorothioate modifications, such as described above, and / or one or more inversion abasic modifications, such as described above. In one embodiment, a gRNA (e.g., sgRNA or dgRNA tracr and / or crRNA), such as any of the above-mentioned gRNA or gRNA components, comprises a phosphorothioate bond between each of the first four 5' residues (e.g., three phosphorothioate bonds included in the 5' ends of one or more polynucleotides, such as consisting thereof), a phosphorothioate bond between each of the first four 3' residues (e.g., three phosphorothioate bonds included in the 5' ends of one or more polynucleotides, such as consisting thereof), a 2'O-methyl modification at each of the first three 5' residues, and a 2'O-methyl modification at each of the first three 3' residues, such as consisting thereof. In one embodiment, a gRNA (e.g., an sgRNA, or tracr and / or crRNA of a dgRNA), e.g., any of the gRNAs or gRNA components described above, comprises, e.g., consists of, a phosphorothioate bond between each of the first four 5' residues (e.g., comprises, e.g., consists of, three phosphorothioate bonds at the 5' end of the one or more polynucleotides), a phosphorothioate bond between each of the first four 3' residues (e.g., comprises, e.g., consists of, three phosphorothioate bonds at the 5' end of the one or more polynucleotides), a 2' O-methyl modification at each of the first three 5' residues, and e.g., consists of, a 2' O-methyl modification at each of the 4th, 3rd, and 2nd 3' residues relative to the end.

[0509] In one embodiment, a gRNA (e.g., an sgRNA, or tracr and / or crRNA of a dgRNA), e.g., any of the gRNAs or gRNA components described above, comprises, e.g., consists of, a phosphorothioate bond between each of the first four 5' residues (e.g., comprises, e.g., consists of, three phosphorothioate bonds at the 5' end of the one or more polynucleotides), a phosphorothioate bond between each of the first four 3' residues (e.g., comprises, e.g., consists of, three phosphorothioate bonds at the 5' end of the one or more polynucleotides), a 2' O-methyl modification at each of the first three 5' residues, a 2' O-methyl modification at each of the first three 3' residues, and an additional inverted abasic residue at each of the 5' and 3' ends.

[0510] In one embodiment, a gRNA (e.g., an sgRNA, or tracr and / or crRNA of a dgRNA), e.g., any of the gRNAs or gRNA components described above, comprises, e.g., consists of, a phosphorothioate bond between each of the first four 5' residues (e.g., comprises, e.g., consists of, three phosphorothioate bonds at the 5' end of the one or more polynucleotides), a phosphorothioate bond between each of the first four 3' residues (e.g., comprises, e.g., consists of, three phosphorothioate bonds at the 5' end of the one or more polynucleotides), a 2' O-methyl modification at each of the first three 5' residues, and a 2' O-methyl modification at each of the 4th, 3rd, and 2nd 3' residues relative to the terminus, and an additional inverted abasic residue at each of the 5' and 3' ends.

[0511] In one embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

[0512] crRNA:

[0513]

[0514] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, e.g., as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini); and

[0515] tracr:

[0516]

[0517] (optionally with inverted abasic residues at the 5' and / or 3' ends).

[0518] In one embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

[0519] crRNA:

[0520]

[0521] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, e.g., as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini); and

[0522] tracr:

[0523]

[0524] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, eg, as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini).

[0525] In one embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

[0526] crRNA:

[0527]

[0528] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, e.g., as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini); and

[0529] tracr:

[0530]

[0531] (optionally with inverted abasic residues at the 5' and / or 3' ends).

[0532] In one embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

[0533] crRNA:

[0534]

[0535] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, e.g., as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini); and

[0536] tracr:

[0537]

[0538] wherein m represents a base with a 2' O-methyl modification and * represents a phosphorothioate linkage (optionally with inverted abasic residues at the 5' and / or 3' termini).

[0539] In one embodiment, the gRNA is a dgRNA and comprises, e.g., consists of:

[0540] crRNA:

[0541] wherein N represents a residue of a targeting domain, e.g., as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini); and

[0542] tracr:

[0543]

[0544] wherein m represents a base with a 2' O-methyl modification and * represents a phosphorothioate linkage (optionally with inverted abasic residues at the 5' and / or 3' termini).

[0545] In one embodiment, the gRNA is an sgRNA and comprises, e.g., consists of:

[0546]

[0547] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, eg, as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini).

[0548] In one embodiment, the gRNA is an sgRNA and comprises, e.g., consists of:

[0549]

[0550] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, eg, as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini).

[0551] In one embodiment, the gRNA is an sgRNA and comprises, e.g., consists of:

[0552]

[0553] wherein m represents a base with a 2' O-methyl modification, * represents a phosphorothioate bond, and N represents a residue of a targeting domain, eg, as described herein (optionally with inverted abasic residues at the 5' and / or 3' termini).

[0554] 6) First Tracr extension

[0555] In the case where the gRNA comprises a first marker stem extension, the tracr can comprise a first tracr extension. The first tracr extension can comprise nucleotides that are complementary to the nucleotides of the first marker stem extension, for example, 80%, 85%, 90%, 95% or 99% complementary, for example, fully complementary. In some aspects, the first tracr extension nucleotides that hybridize to the complementary nucleotides of the first marker stem extension are contiguous. In some aspects, the first tracr extension nucleotides that hybridize to the complementary nucleotides of the first marker stem extension are discontinuous, for example, comprising two or more hybridization regions separated by nucleotides that do not base pair with the nucleotides of the first marker stem extension. In some aspects, the first tracr extension comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some aspects, the first tracr extension comprises SEQ ID NO: 189. In some aspects, the first tracr extension comprises a nucleic acid at least 80%, 85%, 90%, 95%, or 99% homologous to SEQ ID NO: 189.

[0556] Some or all of the nucleotides of the first tracr extension can have modifications, such as those found in Section XIII herein.

[0557] In some embodiments, the sgRNA, from 5' to 3', may comprise, disposed 3' of the targeting domain:

[0558] a)

[0559]

[0560] b)

[0561]

[0562] c)

[0563]

[0564] d)

[0565]

[0566] e) any one of a) to d) above, further comprising at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides at the 3' end, such as 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides;

[0567] f) any one of a) to d) above, further comprising at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides at the 3' end, such as 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides; or

[0568] or g) any one of a) to f) above, further comprising 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 at the 5' end (e.g., at the 5' terminus, e.g., 5' of the targeting domain). In an embodiment, any one of a) to g) above is disposed directly 3' of the targeting domain.

[0569] In one embodiment, the sgRNA of the present invention comprises, for example, consists of, from 5' to 3': [targeting domain]-

[0570]

[0571] In one embodiment, the sgRNA of the present invention comprises, for example, consists of, from 5' to 3': [targeting domain]-

[0572]

[0573] In some embodiments, the dgRNA may comprise:

[0574] crRNA, which comprises from 5' to 3', preferably arranged directly 3' to the targeting domain:

[0575] a) GUUUUAGAGCUA (SEQ ID NO: 182);

[0576] b)GUUUAAGAGCUA (SEQ ID NO: 183);

[0577] c)GUUUUAGAGCUAUGCUG (SEQ ID NO: 199);

[0578] d)GUUUAAGAGCUAUGCUG (SEQ ID NO: 200);

[0579] e) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 201);

[0580] f) GUUUAAGAGCUAUGCUGUUUUG (SEQ ID NO: 202); or

[0581] g)GUUUUAGAGCUAUGCU (SEQ ID NO:226):

[0582] and tracr, which comprises from 5' to 3':

[0583] a)

[0584]

[0585] b)

[0586]

[0587] c)

[0588]

[0589] d)

[0590]

[0591] e)

[0592]

[0593]

[0594] f)

[0595]

[0596] g)

[0597]

[0598] h)

[0599]

[0600] i)

[0601]

[0602] j)

[0603]

[0604] k) any one of a) to j) above, further comprising at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides at the 3′ end, such as 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides;

[0605] l) any one of a) to j) above, further comprising at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides at the 3' end, such as 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides; or

[0606] m) any one of a) to l) above, further comprising at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides at the 5' end (eg, at the 5' terminus), for example, 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides.

[0607] In one embodiment, the sequence of k) above comprises a 3' sequence UUUUUU, for example, if a U6 promoter is used for transcription. In one embodiment, the sequence of k) above comprises a 3' sequence UUUU, for example, if a HI promoter is used for transcription. In one embodiment, the sequence of k) above comprises a variable number of 3'Us, for example, depending on the termination signal of the pol-III promoter used. In one embodiment, the sequence of k) above comprises a variable 3' sequence derived from a DNA template, for example, if a T7 promoter is used. In one embodiment, the sequence of k) above comprises a variable 3' sequence derived from a DNA template, for example, if in vitro transcription is used to produce an RNA molecule. In one embodiment, the sequence of k) above comprises a variable 3' sequence derived from a DNA template, for example, if a pol-II promoter is used to drive transcription.

[0608] In one embodiment, the crRNA comprises, e.g., consists of, a targeting domain and a sequence disposed 3' to the targeting domain (e.g., directly 3' to the targeting domain), the sequence comprising, e.g., consisting of, SEQ ID NO: 201; and the tracr comprises, e.g., consists of:

[0609]

[0610] In one embodiment, the crRNA comprises, e.g., consists of, a targeting domain and a sequence disposed 3' to the targeting domain (e.g., directly disposed 3' to the targeting domain), the sequence comprising, e.g., consisting of, SEQ ID NO: 202; and the tracr comprises, e.g., consists of:

[0611]

[0612] In one embodiment, the crRNA comprises, e.g., consists of, and a sequence disposed 3' to the targeting domain (e.g., directly 3' to the targeting domain), the sequence comprising, e.g., consisting of GUUUUAGAGCUAUGCU (SEQ ID NO: 226); and the tracr comprises, e.g., consists of:

[0613]

[0614] In one embodiment, the crRNA comprises, e.g., consists of, and a sequence disposed 3' to the targeting domain (e.g., directly 3' to the targeting domain), the sequence comprising, e.g., consisting of GUUUUAGAGCUAUGCU (SEQ ID NO: 226); and the tracr comprises, e.g., consists of:

[0615]

[0616] In one embodiment, the crRNA comprises, e.g., consists of, and a sequence disposed 3' to the targeting domain (e.g., directly 3' to the targeting domain), the sequence comprising, e.g., consisting of, GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 201); and tracr comprises, e.g., consists of:

[0617]

[0618] II. gRNA Targeting Domains for the Non-deleted HPFH Region

[0619] The following table provides targeting domains for the non-deleted HPFH region, for use with the gRNA molecules of the present invention, and for use in various aspects of the present invention, for example, to alter the expression of globin genes, such as the fetal hemoglobin gene or the hemoglobin β gene.

[0620] Table 1: gRNA targeting domains for the non-deleted HPFH region. SEQ ID NO: refers to the gRNA targeting domain sequence.

[0621]

[0622]

[0623]

[0624]

[0625] Table 2 below shows those targeting domains that, when included in gRNA molecules according to the methods described in the examples, resulted in at least a 17% increase in fetal hemoglobin (e.g., in erythroid cells differentiated from modified HSPCs) at day 7. gRNA molecules comprising any of these targeting domains are collectively referred to herein as Tier 2 gRNA molecules.

[0626] Table 2 - Targeting domains of hierarchical 2 gRNA molecules

[0627]

[0628]

[0629] Tables 3a and 3b below show those targeting domains that, when included in gRNA molecules according to the methods described in the examples, resulted in the highest increase in fetal hemoglobin (e.g., in erythroid cells differentiated from modified HSPCs) at day 7. gRNA molecules comprising these targeting domains are collectively referred to herein as grade 1 (e.g., grade 1a or grade 1b) gRNA molecules.

[0630] Table 3a - Targeting domains of grade 1a gRNA molecules

[0631] Targeting domain ID GCR-0006 GCR-0008 GCR-0028 GCR-0034 GCR-0048 GCR-0067

[0632] Table 3b.- Targeting domains of grade 1b gRNA molecules

[0633] Targeting domain ID GCR-0001 GCR-0008 GCR-0009 GCR-0010 GCR-0012 GCR-0054

[0634] III. Methods for Designing gRNA

[0635] Methods for designing gRNAs are described herein, including methods for selecting, designing, and verifying target sequences. Exemplary targeting domains are also provided herein. The targeting domains discussed herein can be incorporated into the gRNAs described herein.

[0636] Methods for selecting and validating target sequences and off-target analysis are described, for example, in Mali et al., 2013 SCIENCE 339(6121):823-826; Hsu et al., 2013 NAT BIOTECHNOL, 31(9):827-32; Fu et al., 2014 NAT BIOTECHNOL, doi:10.1038 / nbt.2808. PubMed PM ID:24463574; Heigwer et al., 2014 NAT METHODS 11(2):122-3. doi:10.1038 / nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 BIOINFORMATICS PubMed PMID: 24463181; Xiao A et al., 2014 BIOINFORMATICS PubMed PMID: 24389662.

[0637] For example, software tools can be used to optimize the selection of gRNA in the user's target sequence, such as to minimize the total off-target activity in the entire genome. Off-target activity may not be cutting. For each possible gRNA selection, for example, using Streptococcus pyogenes Cas9, the tool can identify all off-target sequences (such as the aforementioned NAG or NGG PAM) containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of mismatched base pairs in the entire genome. The cutting efficiency at each off-target sequence can be predicted using, for example, an experimentally derived weighted scheme. Each possible gRNA is then arranged according to its predicted total off-target cutting; the top-ranked gRNA representative may have the gRNA with the maximum on-target cutting and the least off-target cutting. Other functions (e.g., automated reagent design for CRISPR construction, primer design for on-target Surveyor determination, and primer design for high-throughput detection and quantitative off-target cutting via next-generation sequencing) can also be included in the tool. Candidate gRNA molecules can be evaluated by methods known in the art or as described herein.

[0638] Although software algorithms can be used to generate an initial list of potential gRNA molecules, cutting efficiency and specificity do not necessarily reflect predicted values, and gRNA molecules generally need to be screened in specific cell lines (e.g., primary human cell lines, such as human HSPCs, such as human CD34+ cells) to determine, for example, changes in cutting efficiency, insertion / deletion formation, cutting specificity, and desired phenotypes. These properties can be determined by the methods described herein.

[0639] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0001 (SEQ ID NO: 1, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0640] sgRNA GCR-0001 #1:

[0641]

[0642] sgRNA GCR-0001 #2:

[0643]

[0644] sgRNA GCR-0001 #3:

[0645]

[0646] dgRNA GCR-0001 #1:

[0647] crRNA:

[0648] tracr:

[0649]

[0650] dgRNA GCR-0001#2:

[0651] crRNA:

[0652] tracr:

[0653]

[0654] dgRNA GCR-0001#3:

[0655] crRNA:

[0656] tracr:

[0657]

[0658] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0659] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0006 (SEQ ID NO: 6, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0660] sgRNA GCR-0006 #1:

[0661]

[0662]

[0663] sgRNA GCR-0006#2:

[0664]

[0665] sgRNA GCR-0006#3:

[0666]

[0667] dgRNA GCR-0006#1:

[0668] crRNA:

[0669] tracr:

[0670]

[0671] dgRNA GCR-0006#2:

[0672] crRNA:

[0673] tracr:

[0674]

[0675] dgRNA GCR-0006#3:

[0676] crRNA:

[0677] tracr:

[0678]

[0679] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0680] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0008 (SEQ ID NO: 8, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0681] sgRNA GCR-0008#1:

[0682]

[0683] sgRNA GCR-0008 #2:

[0684]

[0685] sgRNA GCR-0008 #3:

[0686]

[0687] dgRNA GCR-0008#1:

[0688] crRNA:

[0689] tracr:

[0690]

[0691] dgRNA GCR-0008#2:

[0692] crRNA:

[0693] tracr:

[0694]

[0695] dgRNA GCR-0008#3:

[0696] crRNA:

[0697] tracr:

[0698]

[0699] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0700] In aspects of the invention, a gRNA comprising the targeting domain of GCR-009 (SEQ ID NO: 9, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0701] sgRNA GCR-0009#1:

[0702]

[0703] sgRNA GCR-0009#2:

[0704]

[0705] sgRNA GCR-0009#3:

[0706]

[0707] dgRNA GCR-0009#1:

[0708] crRNA:

[0709] tracr:

[0710]

[0711] dgRNA GCR-0009#2:

[0712] crRNA:

[0713] tracr:

[0714]

[0715] dgRNA GCR-0009#3:

[0716] crRNA:

[0717] tracr:

[0718]

[0719] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0720] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0010 (SEQ ID NO: 10, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0721] sgRNA GCR-0010#1:

[0722]

[0723] sgRNA GCR-0010#2:

[0724]

[0725] sgRNA GCR-0010#3:

[0726]

[0727] dgRNA GCR-0010#1:

[0728] crRNA:

[0729] tracr:

[0730]

[0731] dgRNA GCR-0010#2:

[0732] crRNA:

[0733] tracr:

[0734]

[0735] dgRNA GCR-0010#3:

[0736] crRNA:

[0737] tracr:

[0738]

[0739] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0740] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0011 (SEQ ID NO: 11, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0741] sgRNA GCR-0011 #1:

[0742]

[0743] sgRNA GCR-0011#2:

[0744]

[0745] sgRNA GCR-0011#3:

[0746]

[0747] dgRNA GCR-0011#1:

[0748] crRNA:

[0749] tracr:

[0750]

[0751] dgRNA GCR-0011#2:

[0752] crRNA:

[0753] tracr:

[0754]

[0755] dgRNA GCR-0011#3:

[0756]

[0757] tracr:

[0758]

[0759] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0760] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0012 (SEQ ID NO: 12, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0761] sgRNA GCR-0012 #1:

[0762]

[0763] sgRNA GCR-0012 #2:

[0764]

[0765] sgRNA GCR-0012#3:

[0766]

[0767] dgRNA GCR-0012#1:

[0768] crRNA:

[0769] tracr:

[0770]

[0771] dgRNA GCR-0012#2:

[0772] crRNA:

[0773] tracr:

[0774]

[0775] dgRNA GCR-0012 #3:

[0776] crRNA:

[0777] tracr:

[0778]

[0779] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0780] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0028 (SEQ ID NO: 28, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0781] sgRNA GCR-0028 #1:

[0782]

[0783] sgRNA GCR-0028#2:

[0784]

[0785]

[0786] sgRNAGCR-0028 #3:

[0787]

[0788] dgRNAGCR-0028#1:

[0789] crRNA:

[0790] tracr:

[0791]

[0792] dgRNAGCR-0028#2:

[0793] crRNA:

[0794] tracr:

[0795]

[0796] dgRNAGCR-0028#3:

[0797] crRNA:

[0798] tracr:

[0799]

[0800] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0801] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0034 (SEQ ID NO: 34, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0802] sgRNA GCR-0034#1:

[0803]

[0804] sgRNA GCR-0034#2:

[0805]

[0806] sgRNA GCR-0034 #3:

[0807]

[0808] dgRNA GCR-0034#1:

[0809] crRNA:

[0810] tracr:

[0811]

[0812] dgRNA GCR-0034#2:

[0813] crRNA:

[0814] tracr:

[0815]

[0816] dgRNAGCR-0034#3:

[0817] crRNA:

[0818] tracr:

[0819]

[0820] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0821] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0045 (SEQ ID NO: 45, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0822] sgRNAGCR-0045#1:

[0823]

[0824] sgRNAGCR-0045#2:

[0825]

[0826] sgRNA GCR-0045#3:

[0827]

[0828] dgRNAGCR-0045#1:

[0829] crRNA:

[0830] tracr:

[0831]

[0832] dgRNAGCR-0045#2:

[0833] crRNA:

[0834] tracr:

[0835]

[0836] dgRNA GCR-0045#3:

[0837] crRNA:

[0838] tracr:

[0839]

[0840] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0841] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0046 (SEQ ID NO: 46, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0842] sgRNAGCR-0046 #1:

[0843]

[0844] sgRNAGCR-0046 #2:

[0845]

[0846] sgRNAGCR-0046#3:

[0847]

[0848] dgRNAGCR-0046#1:

[0849] crRNA:

[0850] tracr:

[0851]

[0852] dgRNAGCR-0046#2:

[0853] crRNA:

[0854] tracr:

[0855]

[0856] dgRNAGCR-0046#3:

[0857] crRNA:

[0858] tracr:

[0859]

[0860] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0861] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0047 (SEQ ID NO: 47, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0862] sgRNA GCR-0047 #1:

[0863]

[0864] sgRNA GCR-0047#2:

[0865]

[0866] sgRNAGCR-0047 #3:

[0867]

[0868] dgRNA GCR-0047 #1:

[0869] crRNA:

[0870] tracr:

[0871]

[0872] dgRNAGCR-0047#2:

[0873] crRNA:

[0874] tracr:

[0875]

[0876] dgRNAGCR-0047#3:

[0877] crRNA:

[0878] tracr:

[0879]

[0880] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0881] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0048 (SEQ ID NO: 48, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0882] sgRNAGCR-0048#1:

[0883]

[0884] sgRNAGCR-0048#2:

[0885]

[0886] sgRNAGCR-0048 #3:

[0887]

[0888] dgRNA GCR-0048#1:

[0889] crRNA:

[0890] tracr:

[0891]

[0892] dgRNA GCR-0048#2:

[0893] crRNA:

[0894] tracr:

[0895]

[0896] dgRNA GCR-0048#3:

[0897] crRNA:

[0898] tracr:

[0899]

[0900] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0901] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0050 (SEQ ID NO: 50, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0902] sgRNA GCR-0050 #1:

[0903]

[0904] sgRNA GCR-0050 #2:

[0905]

[0906] sgRNAGCR-0050 #3:

[0907]

[0908] dgRNA GCR-0050 #1:

[0909] crRNA:

[0910] tracr:

[0911]

[0912] dgRNA GCR-0050#2:

[0913] crRNA:

[0914] tracr:

[0915]

[0916] dgRNAGCR-0050#3:

[0917] crRNA:

[0918] tracr:

[0919]

[0920] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0921] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0051 (SEQ ID NO: 51, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0922] sgRNA GCR-0051#1:

[0923]

[0924] sgRNA GCR-0051#2:

[0925]

[0926] sgRNAGCR-0051 #3:

[0927]

[0928] dgRNAGCR-0051 #1:

[0929] crRNA:

[0930] tracr:

[0931]

[0932]

[0933] dgRNA GCR-0051 #2:

[0934] crRNA:

[0935] tracr:

[0936]

[0937] dgRNAGCR-0051#3:

[0938] crRNA:

[0939] tracr:

[0940]

[0941] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0942] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0053 (SEQ ID NO: 53, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0943] sgRNA GCR-0053#1:

[0944]

[0945] sgRNAGCR-0053#2:

[0946]

[0947] sgRNAGCR-0053 #3:

[0948]

[0949] dgRNA GCR-0053#1:

[0950] crRNA:

[0951] tracr:

[0952]

[0953] dgRNA GCR-0053#2:

[0954] crRNA:

[0955] tracr:

[0956]

[0957] dgRNAGCR-0053#3:

[0958] crRNA:

[0959] tracr:

[0960]

[0961] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0962] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0054 (SEQ ID NO: 54, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0963] sgRNA GCR-0054#1:

[0964]

[0965] sgRNA GCR-0054 #2:

[0966]

[0967] sgRNA GCR-0054 #3:

[0968]

[0969] dgRNA GCR-0054#1:

[0970] crRNA:

[0971] tracr:

[0972]

[0973] dgRNA GCR-0054#2:

[0974] crRNA:

[0975] tracr:

[0976]

[0977] dgRNA GCR-0054#3:

[0978] crRNA:

[0979] tracr:

[0980]

[0981] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[0982] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0058 (SEQ ID NO: 58, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[0983] sgRNA GCR-0058 #1:

[0984]

[0985] sgRNA GCR-0058#2:

[0986]

[0987] sgRNA GCR-0058#3:

[0988]

[0989] dgRNA GCR-0058#1:

[0990] crRNA:

[0991] tracr:

[0992]

[0993] dgRNA GCR-0058#2:

[0994] crRNA:

[0995] tracr:

[0996]

[0997] dgRNA GCR-0058#3:

[0998] crRNA:

[0999] tracr:

[1000]

[1001] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[1002] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0062 (SEQ ID NO: 62, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[1003] sgRNA GCR-0062 #1:

[1004]

[1005] sgRNA GCR-0062 #2:

[1006]

[1007] sgRNA GCR-0062 #3:

[1008]

[1009] dgRNA GCR-0062#1:

[1010] crRNA:

[1011] tracr:

[1012]

[1013] dgRNA GCR-0062#2:

[1014] crRNA:

[1015] tracr:

[1016]

[1017] dgRNA GCR-0062#3:

[1018] crRNA:

[1019] tracr:

[1020]

[1021] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[1022] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0063 (SEQ ID NO: 63, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[1023] sgRNA GCR-0063#1:

[1024]

[1025] sgRNA GCR-0063#2:

[1026]

[1027] sgRNA GCR-0063 #3:

[1028]

[1029] dgRNA GCR-0063#1:

[1030] crRNA:

[1031] tracr:

[1032]

[1033] dgRNA GCR-0063#2:

[1034] crRNA:

[1035] tracr:

[1036]

[1037] dgRNA GCR-0063#3:

[1038] crRNA:

[1039] tracr:

[1040]

[1041] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[1042] In aspects of the invention, a gRNA comprising the targeting domain of GCR-0067 (SEQ ID NO: 67, the unmodified sequence is underlined), such as one of the gRNA molecules described below, can be used in the CRISPR systems, methods, cells, and other aspects and embodiments of the invention, including in aspects involving more than one gRNA molecule, such as those described herein:

[1043] sgRNA GCR-0067#1:

[1044]

[1045] sgRNA GCR-0067#2:

[1046]

[1047] sgRNA GCR-0067#3:

[1048]

[1049] dgRNA GCR-0067#1:

[1050] crRNA:

[1051] tracr:

[1052]

[1053] dgRNA GCR-0067#2:

[1054] crRNA:

[1055] tracr:

[1056]

[1057] dgRNA GCR-0067#3:

[1058] crRNA:

[1059] tracr:

[1060]

[1061] In each of the above-mentioned gRNA molecules, "*" represents a phosphorothioate bond between adjacent nucleotides, and "mN" (wherein N = A, G, C or U) represents a 2'-OMe modified nucleotide. In an embodiment, any gRNA molecule described herein (e.g., as described above) is compounded with a Cas9 molecule (e.g., as described herein) to form a ribonucleoprotein complex (RNP). Such RNPs are particularly useful in the methods, cells, and other aspects and embodiments of the present invention (e.g., as described herein).

[1062] IV. Cas molecules

[1063] Cas9 molecule

[1064] In a preferred embodiment, the Cas molecule is a Cas9 molecule. Cas9 molecules of various species can be used in the methods and compositions described herein. Although the Streptococcus pyogenes Cas9 molecule is the subject of most of the disclosures herein, Cas9 molecules derived from or based on the Cas9 proteins of other species listed herein can also be used. In other words, other Cas9 molecules (e.g., Streptococcus thermophilus, Staphylococcus aureus, and / or Neisseria meningitidis Cas9 molecules) can be used in the systems, methods, and compositions described herein. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces species, Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides species, Blastopirellula marina, Bradyrhizobium species, Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridiu cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacler diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensiscanadensis), Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae, Methylosinus species, Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria species, Neisseria wadsworthii, Nitrosomonas species, Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodopseudomonas species, Simonsiella muelleri, Sphingomonas species, Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus species, Subdoligranulum sp., Tislrella mobilis, Treponema species, or Verminephrobacter eiseniae.

[1065] A Cas9 molecule (as the term is used herein) refers to a molecule that can interact with a gRNA molecule (e.g., a sequence of a tracr domain) and, together with the gRNA molecule, localize (e.g., target or home) to a site comprising a target sequence and a PAM sequence.

[1066] In one embodiment, the Cas9 molecule is capable of cutting a target nucleic acid molecule, which may be referred to herein as an active Cas9 molecule. In one embodiment, the active Cas9 molecule comprises one or more of the following activities: nickase activity, i.e., the ability to cut a single strand (e.g., a non-complementary strand or a complementary strand) of a nucleic acid molecule; double-stranded nuclease activity, i.e., the ability to cut two strands of a double-stranded nucleic acid and produce a double-strand break, which in one embodiment is in the presence of two nickase activities; endonuclease activity; exonuclease activity; and helicase activity, i.e., the ability to unravel the helical structure of a double-stranded nucleic acid.

[1067] In one embodiment, the enzymatically active Cas9 molecule cuts two DNA chains and causes double-strand breaks. In one embodiment, the Cas9 molecule cuts only one chain, such as the chain hybridized with the gRNA or the chain complementary to the chain hybridized with the gRNA. In one embodiment, the active Cas9 molecule comprises a cleavage activity associated with an HNH-like domain. In one embodiment, the active Cas9 molecule comprises a cleavage activity associated with an N-terminal RuvC-like domain. In one embodiment, the active Cas9 molecule comprises a cleavage activity associated with an HNH-like domain and a cleavage activity associated with an N-terminal RuvC-like domain. In one embodiment, the active Cas9 molecule comprises an HNH-like domain with activity or cleavage capability and an N-terminal RuvC-like domain with inactivity or no cleavage capability. In one embodiment, the active Cas9 molecule comprises an HNH-like domain with inactivity or no cleavage capability and an N-terminal RuvC-like domain with activity or cleavage capability.

[1068] In one embodiment, the ability of active Cas9 molecules to interact with target nucleic acids and cut target nucleic acids is PAM sequence dependent. The PAM sequence is a sequence in the target nucleic acid. In one embodiment, the cutting of the target nucleic acid occurs upstream of the PAM sequence. Active Cas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In one embodiment, the active Cas9 molecule of Streptococcus pyogenes recognizes the sequence motif NGG and guides the cutting of the target nucleic acid sequence 1 to 10 (e.g., 3 to 5) base pairs upstream of the sequence. See, for example, Mali et al., SCIENCE [Science] 2013; 339 (6121): 823-826. In one embodiment, the active Cas9 molecule of Streptococcus thermophilus recognizes the sequence motifs NGGNG and NNAGAAW (W=A or T) and guides the cutting of the core target nucleic acid sequence 1 to 10 (e.g., 3 to 5) base pairs upstream of these sequences. See, e.g., Horvath et al., SCIENCE 2010; 327(5962): 167-170; and Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In one embodiment, an active Cas9 molecule from Streptococcus mutans (S. mutans) recognizes the sequence motif NGG or NAAR (RA or G) and directs cleavage of the core target nucleic acid sequence 1 to 10 (e.g., 3 to 5) base pairs upstream of the sequence. See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400.

[1069] In one embodiment, the active Cas9 molecule of Staphylococcus aureus recognizes sequence motif NNGRR (R=A or G) and guides the cutting of the target nucleic acid sequence 1 to 10 (e.g., 3 to 5) base pairs upstream of the sequence. See, for example, Ran F. et al., NATURE [Nature], Vol. 520, 2015, pp. 186-191. In one embodiment, the active Cas9 molecule of Neisseria meningitidis recognizes sequence motif NNNNGATT and guides the cutting of the target nucleic acid sequence 1 to 10 (e.g., 3 to 5) base pairs upstream of the sequence. See, for example, Hou et al., PNAS EARLY EDITION [Early Edition of the Proceedings of the National Academy of Sciences of the United States] 2013, 1 -6. The ability of the Cas9 molecule to recognize the PAM sequence can be determined, for example, using the conversion assay described in Jinek et al., SCIENCE [Science] 2012, 337:816.

[1070] Some Cas9 molecules have the ability to interact with gRNA molecules and work together with gRNA molecules to home (e.g., target or locate) to the core target domain, but cannot cut the target nucleic acid, or cannot cut at an effective rate. Cas9 molecules with no or substantially no cutting activity may be referred to herein as inactive Cas9 (enzymatically inactivated Cas9), dead Cas9 or dCas9 molecules. For example, as measured by an assay as described herein, an inactive Cas9 molecule may lack cutting activity or have substantially less cutting activity, for example, less than 20%, 10%, 5%, 1% or 0.1% of the cutting activity of a reference Cas9 molecule.

[1071] Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013; 10: 5, 727-737. Such Cas9 molecules include cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, cluster 7 bacterial family, cluster 8 bacterial family, cluster 9 bacterial family, cluster 10 bacterial family, cluster 11 bacterial family, cluster 12 bacterial family, cluster 13 bacterial family, cluster 14 bacterial family, cluster 15 bacterial family, cluster 16 bacterial family, cluster 17 bacterial family, cluster 18 bacterial family, cluster 19 bacterial family, cluster 20 bacterial family, cluster 21 bacterial family, cluster 22 bacterial family, cluster 23 bacterial family, cluster 24 bacterial family, cluster 25 bacterial family, cluster 26 bacterial family, cluster 27 bacterial family, cluster 28 bacterial family, cluster 29 bacterial family, cluster 30 bacterial family, cluster 31 bacterial family, cluster 32 bacterial family, cluster 33 bacterial family, cluster 34 bacterial family, cluster 35 bacterial family, cluster 36 bacterial family, cluster 37 bacterial family, cluster 38 bacterial family, cluster 39 bacterial family, cluster 40 bacterial family, cluster 41 bacterial family, cluster 42 bacterial family, cluster 43 bacterial family, cluster 44 bacterial family, cluster 45 bacterial family, cluster 46 bacterial family, cluster 47 bacterial family, cluster 48 bacterial family, cluster 49 bacterial family, cluster 50 bacterial family, cluster 51 bacterial family, cluster 52 bacterial family, cluster 53 bacterial family, cluster 54 bacterial family, cluster 55 bacterial family, cluster 56 bacterial family, cluster 57 bacterial family, cluster 58 bacterial family, cluster 59 bacterial family, cluster 60 bacterial family, cluster 61 bacterial family, cluster 62 bacterial family, cluster 63 bacterial family, cluster 64 bacterial family, cluster 65 bacterial family, cluster 66 bacterial family, cluster 67 bacterial family, cluster 68 bacterial family, cluster 69 bacterial family, cluster 70 bacterial family, cluster 71 bacterial family, cluster 72 bacterial family, cluster 73 bacterial family, cluster 7 Cas9 molecules from a bacterial family, cluster 41 bacterial family, cluster 42 bacterial family, cluster 43 bacterial family, cluster 44 bacterial family, cluster 45 bacterial family, cluster 46 bacterial family, cluster 47 bacterial family, cluster 48 bacterial family, cluster 49 bacterial family, cluster 50 bacterial family, cluster 51 bacterial family, cluster 52 bacterial family, cluster 53 bacterial family, cluster 54 bacterial family, cluster 55 bacterial family, cluster 56 bacterial family, cluster 57 bacterial family, cluster 58 bacterial family, cluster 59 bacterial family, cluster 60 bacterial family, cluster 61 bacterial family, cluster 62 bacterial family, cluster 63 bacterial family, cluster 64 bacterial family, cluster 65 bacterial family, cluster 66 bacterial family, cluster 67 bacterial family, cluster 68 bacterial family, cluster 69 bacterial family, cluster 70 bacterial family, cluster 71 bacterial family, cluster 72 bacterial family, cluster 73 bacterial family, cluster 74 bacterial family, cluster 75 bacterial family, cluster 76 bacterial family, cluster 77 bacterial family, or cluster 78 bacterial family.

[1072] Exemplary naturally occurring Cas9 molecules include Cas9 molecules of the bacterial family Cluster 1. Examples include the Cas9 molecules of Streptococcus pyogenes (e.g., strains SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131, and SSI-1), Streptococcus thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), Streptococcus mutans (e.g., strains UA 159, NN2025), S. macacae (e.g., strain NCTC1 1558), S. gallolylicus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS124), S. dysgalactiae (e.g., strain GGS124), S. bovis (e.g., strain ATCC 700338), S. cmginosus (e.g., strain F021 1), S. agalactiae (e.g., strains NEM316, A909), Listeria monocytogenes (e.g., strain F6854), L. innocua (e.g., strain Clip 1 1262), E. italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strains 1,231, 408). Additional exemplary Cas9 molecules are the Cas9 molecule from Neisseria meningitidis (Hou et al. PNAS Early Edition 2013, 1-6) and the Cas9 molecule from Staphylococcus aureus.

[1073] In one embodiment, the Cas9 molecule (e.g., an active Cas9 molecule or an inactive Cas9 molecule) comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence (e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA Biology 2013, 10:5, T2T-T, 1; Hou et al. PNAS Early Edition 2013, 1-6); or to any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence (e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA Biology 2013, 10:5, T2′I-T, 1; Hou et al. PNAS Early Edition 2013, 1-6). Early Edition [PNAS Early Edition] 2013, 1-6) that differs by no more than 1%, 2%, 5%, 10%, 15%, 20%, 30% or 40% of amino acid residues from any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence (e.g., from a species listed herein or described in Chylinski et al., RNA Biology [RNA Biology] 2013, 10:5, 'I2'I-T, 1; Hou et al. PNAS Early Edition [PNAS Early Edition] 2013, 10:5, 'I2'I-T, 1; Hou et al. PNAS Early Edition [PNAS Early Edition] 2013, 10:5, 'I2'I-T, 1; Hou et al. PNAS Early Edition [PNAS Early Edition] 2013, 10: Edition [Proceedings of the National Academy of Sciences of the United States of America, Early Edition] 2013, 1-6) differs by at least 1, 2, 5, 10 or 20 amino acids but does not differ by more than 100, 80, 70, 60, 50, 40 or 30 amino acids; or an amino acid sequence that is identical to any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence (e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA Biology [RNA Biology] 2013, 10:5, 'I2'I-T, 1; Hou et al., PNAS Early Edition [Proceedings of the National Academy of Sciences of the United States of America, Early Edition] 2013, 1-6).

[1074] In one embodiment, the Cas9 molecule comprises an amino acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to S. pyogenes Cas9; an amino acid sequence that differs by no more than 1%, 2%, 5%, 10%, 15%, 20%, 30% or 40% of the amino acid residues when compared to S. pyogenes Cas9; an amino acid sequence that differs by at least 1, 2, 5, 10 or 20 amino acids but no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from S. pyogenes Cas9; or an amino acid sequence that is identical to S. pyogenes Cas9:

[1075]

[1076]

[1077]

[1078]

[1079]

[1080] In an embodiment, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO:205, which includes one or more mutations to positively charged amino acids (e.g., lysine, arginine, or histidine), which introduce uncharged or non-polar amino acids (e.g., alanine) at the position. In an embodiment, the mutation is for one or more positively charged amino acids in the nt groove of Cas9. In an embodiment, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO:205, which includes a mutation at SEQ ID NO:205 position 855, such as at SEQ ID NO:205 position 855 is mutated to uncharged amino acid, such as alanine. In an embodiment, the Cas9 molecule is only at SEQ ID NO:205 position 855 relative to SEQ ID NO:205 has a mutation, such as a mutation to uncharged amino acid, such as alanine. In embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO: 205 comprising a mutation at position 810, a mutation at position 1003, and / or a mutation at position 1060 of SEQ ID NO: 205, e.g., a mutation to alanine at position 810, position 1003, and / or position 1060 of SEQ ID NO: 205. In embodiments, the Cas9 molecule has mutations only at position 810, position 1003, and position 1060 of SEQ ID NO: 205 relative to SEQ ID NO: 205, e.g., wherein each mutation is a mutation to an uncharged amino acid, e.g., alanine. In an embodiment, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO: 205 comprising 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 to alanine at position 848, position 1003, and / or position 1060 of SEQ ID NO: 205. In an embodiment, the Cas9 molecule has mutations only at position 848, position 1003, and position 1060 of SEQ ID NO: 205 relative to SEQ ID NO: 205, e.g., wherein each mutation is a mutation to an uncharged amino acid, e.g., alanine. In an embodiment, the Cas9 molecule is a Cas9 molecule as described in Slaymaker et al., Science Express, available on December 1, 2015 at Science DOI: 10.1126 / science.aad5227.

[1081] In an embodiment, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO:205, comprising one or more mutations. In an embodiment, the Cas9 variant is included in the mutation at SEQ ID NO:205 position 80, for example, comprising SEQ ID NO:205 position 80 leucine (i.e., comprising SEQ ID NO:205 with C80L mutation at leucine (i.e., comprising SEQ ID NO:205 with C574E mutation at leucine (i.e., comprising SEQ ID NO:205 with C574E mutation at leucine (i.e., comprising SEQ ID NO:205 with C80L ... Without being bound by theory, it is believed that such mutations improve the solubility properties of the Cas9 molecule.

[1082] In an embodiment, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO: 205 comprising one or more mutations. In an embodiment, the Cas9 variant is included in a mutation at position 147 of SEQ ID NO: 205, e.g., comprising a tyrosine at position 147 of SEQ ID NO: 205 (i.e., comprising, e.g., consisting of, a SEQ ID NO: 205 having a D147Y mutation). In an embodiment, the Cas9 variant is included in a mutation at position 411 of SEQ ID NO: 205, e.g., comprising a threonine at position 411 of SEQ ID NO: 205 (i.e., comprising, e.g., consisting of, a SEQ ID NO: 205 having a P411T mutation). In an embodiment, the Cas9 variant comprises a mutation at position 147 of SEQ ID NO: 205 and a mutation at position 411, e.g., comprising a tyrosine at position 147 of SEQ ID NO: 205 and a threonine at position 411 of SEQ ID NO: 205 (i.e., comprising, e.g., consisting of, SEQ ID NO: 205 having a D147Y mutation and a P411T mutation). Without being bound by theory, it is believed that such mutations improve the targeting efficiency of the Cas9 molecule, e.g., in yeast.

[1083] In an embodiment, the Cas9 molecule is a Streptococcus pyogenes Cas9 variant of SEQ ID NO: 205 comprising one or more mutations. In an embodiment, the Cas9 variant comprises a mutation at position 1135 of SEQ ID NO: 205, e.g., comprising a glutamic acid at position 1135 of SEQ ID NO: 205 (i.e., comprising, e.g., consisting of, SEQ ID NO: 205 having a D1135E mutation). Without being bound by theory, it is believed that such mutations improve the selectivity of the Cas9 molecule for NGG PAM sequences relative to NAG PAM sequences.

[1084] In an embodiment, Cas9 molecule is SEQ ID NO:205 streptococcus pyogenes Cas9 variant, it includes one or more mutations, and these mutations introduce uncharged or non-polar amino acid (such as alanine) in some position.In an embodiment, Cas9 molecule is SEQ ID NO:205 streptococcus pyogenes Cas9 variant, it is included in SEQ ID NO:205 position 497 mutation, position 661 mutation, position 695 mutation and / or position 926 mutation, for example, at SEQ ID NO:205 position 497, position 661, position 695 and / or position 926 are mutated to alanine.In an embodiment, Cas9 molecule is only at SEQ ID NO:205 position 497, position 661, position 695 and position 926 relative to SEQ ID NO:205 has mutation, for example, wherein each mutation is to mutate to uncharged amino acid, such as alanine.Not being bound by theory, it is believed that such mutation reduces the cutting of Cas9 molecule at off-target site.

[1085] It will be understood that the mutations described herein for the Cas9 molecules can be combined and can be combined with any of the fusions or other modifications described herein and the Cas9 molecules tested in the assays described herein.

[1086] Various types of Cas molecules can be used to practice the invention disclosed herein. In some embodiments, Cas molecules of type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, type I or type III Cas molecules can be used. Exemplary Cas molecules (and Cas systems) are described, for example, in Haft et al., PLoS COMPUTATIONAL BIOLOGY [Public Library of Science Computational Biology] 2005, 1(6): e60 and Makarova et al., NATURE REVIEW MICROBIOLOGY [Nature Microbiology Review] 201 1, 9: 467-477, the contents of which are incorporated herein by reference in their entirety.

[1087] In one embodiment, the Cas9 molecule comprises one or more of the following activities: nickase activity; double-stranded cleavage activity (e.g., endonuclease and / or exonuclease activity); helicase activity; or the ability to localize to a target nucleic acid together with a gRNA molecule.

[1088] Altered Cas9 molecules

[1089] Naturally occurring Cas9 molecules have many properties, including: nickase activity; nuclease activity (e.g., endonuclease and / or exonuclease activity); helicase activity; the ability to functionally associate with gRNA molecules; and the ability to target (or localize to) sites on nucleic acids (e.g., PAM recognition and specificity). In one embodiment, the Cas9 molecule may include all or a subset of these properties. In a typical embodiment, the Cas9 molecule has the ability to interact with the gRNA molecule and localize to sites in the nucleic acid together with the gRNA molecule. Other activities (e.g., PAM specificity, cleavage activity, or helicase activity) may vary more widely in the Cas9 molecule.

[1090] Cas9 molecules with desired properties can be made in a variety of ways, for example, by altering a parent (e.g., a naturally occurring Cas9 molecule) to provide an altered Cas9 molecule with desired properties. For example, one or more mutations or differences relative to the parent Cas9 molecule can be introduced. Such mutations and differences include: substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In one embodiment, the Cas9 molecule can comprise one or more mutations or differences relative to a reference Cas9 molecule, for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mutations but less than 200, 100, or 80 mutations.

[1091] In one embodiment, one or more mutations have no substantial effect on Cas9 activity (e.g., Cas9 activity as described herein). In one embodiment, one or more mutations have a substantial effect on Cas9 activity (e.g., Cas9 activity as described herein). In one embodiment, exemplary activity includes one or more of PAM specificity, cleavage activity, and helicase activity. One or more mutations may, for example, be present in the following: one or more RuvC-like domains, such as an N-terminal RuvC-like domain; an HNH-like domain; a region outside a RuvC-like domain and an HNH-like domain. In some embodiments, one or more mutations are present in an N-terminal RuvC-like domain. In some embodiments, one or more mutations are present in an HNH-like domain. In some embodiments, mutations are present in both an N-terminal RuvC-like domain and an HNH-like domain.

[1092] Whether a particular sequence (e.g., a substitution) can affect one or more activities, such as targeting activity, cleavage activity, etc., can be assessed or predicted, for example, by assessing whether the mutation is conservative or by the methods described in Section III. In one embodiment, a "non-essential" amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule (e.g., a naturally occurring Cas9 molecule, e.g., an active Cas9 molecule) without eliminating or, more preferably, substantially altering the Cas9 activity (e.g., cleavage activity), whereas altering an "essential" amino acid residue results in a significant loss of activity (e.g., cleavage activity).

[1093] Cas9 molecules with altered PAM recognition or no PAM recognition

[1094] Naturally occurring Cas9 molecules can recognize specific PAM sequences, such as the PAM recognition sequences described above for Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Staphylococcus aureus, and Neisseria meningitidis.

[1095] 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 PAM specificity that is unrelated to the naturally occurring Cas9 molecule, or a PAM specificity that is unrelated to the naturally occurring Cas9 molecule with which it has the closest sequence homology. For example, the naturally occurring Cas9 molecule can be altered, for example, to change PAM recognition, for example, to change the PAM sequence recognized by the Cas9 molecule to reduce off-target sites and / or improve specificity; or to eliminate the PAM recognition requirement. In one embodiment, the Cas9 molecule can be altered, for example, to increase the length of the PAM recognition sequence and / or to increase the Cas9 specificity to a high level of identity to reduce off-target sites and increase specificity. In one embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Directed evolution can be used to generate Cas9 molecules that recognize different PAM sequences and / or have reduced off-target activity. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described in, for example, Esvelt et al., Nature 2011, 472(7344):499-503. Candidate Cas9 molecules can be evaluated, for example, by the methods described herein.

[1096] Non-cutting and modified cutting Cas9 molecules

[1097] In one embodiment, the Cas9 molecule includes a cutting property different from a naturally occurring Cas9 molecule (e.g., different from a naturally occurring Cas9 molecule with closest homology). For example, the Cas9 molecule may be different from a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of Streptococcus pyogenes), and the following ability may be eliminated: for example, compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of Streptococcus pyogenes), it regulates (e.g., reduces or increases) the ability (endonuclease and / or exonuclease activity) of cutting of a double-strand break; for example, compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of Streptococcus pyogenes), it regulates (e.g., reduces or increases) the ability (nickase activity) of cutting of a single nucleic acid chain (e.g., a non-complementary chain of a nucleic acid molecule or a complementary chain of a nucleic acid molecule); or the ability to cut a nucleic acid molecule (e.g., a double-stranded or single-stranded nucleic acid molecule).

[1098] Modified cleavage-active Cas9 molecules

[1099] In one embodiment, the active Cas9 molecule comprises one or more of the following activities: cleavage activity associated with the N-terminal RuvC-like domain; cleavage activity associated with the HNH-like domain; cleavage activity associated with the HNH domain and cleavage activity associated with the N-terminal RuvC-like domain.

[1100] In one embodiment, the Cas9 molecule is a Cas9 nickase, e.g., cleaving only a single strand of DNA. In one embodiment, the Cas9 nickase comprises a mutation at position 10 of SEQ ID NO: 205 and / or a mutation at position 840, e.g., comprising a D10A and / or H840A mutation to SEQ ID NO: 205.

[1101] Non-cutting, inactive Cas9 molecules

[1102] In one embodiment, the altered Cas9 molecule is an inactive Cas9 molecule that does not cut nucleic acid molecules (double-stranded or single-stranded nucleic acid molecules) or cuts nucleic acid molecules with a significantly lower efficiency (e.g., less than 20%, 10%, 5%, 1% or 0.1% of the cutting activity of the reference Cas9 molecule, for example, as measured by an assay as described herein). The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, such as a naturally occurring Cas9 molecule, such as a Cas9 molecule of Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitidis. In one embodiment, the reference Cas9 molecule is a naturally occurring Cas9 molecule with the closest sequence identity or homology. In one embodiment, the inactive Cas9 molecule lacks significant cutting activity associated with the N-terminal RuvC-like domain and cutting activity associated with the HNH-like domain.

[1103] In one embodiment, the Cas9 molecule is dCas9. Tsai et al. (2014), Nat. Biotech. 32: 569-577.

[1104] The catalytically inactivated Cas9 molecule can be fused with a transcriptional repressor. The inactive Cas9 fusion protein is compounded with the gRNA and positioned at the DNA sequence specified by the gRNA targeting domain, but unlike active Cas9, it does not cut the target DNA. The fusion of the effector domain (such as the transcription repression domain) and the inactive Cas9 makes it possible to recruit the effector to any DNA site specified by the gRNA. The Cas9 fusion protein site-specific targeting to the promoter region of the gene can block or affect the binding of the polymerase to the promoter region, for example, Cas9 is fused to a transcription factor (such as a transcription activator) and / or a transcription enhancer is bound to nucleic acid to increase or inhibit transcriptional activation. Alternatively, the fusion site-specific targeting of Cas9 to a transcriptional repressor to the promoter region of the gene can be used to reduce transcriptional activation.

[1105] Transcriptional repressors or transcriptional repressor domains that can be fused to the inactive Cas9 molecule can include a ruppel-associated box (KRAB or SKD), a Mad mSIN3 interacting domain (SID), or an ERF repressor domain (ERD).

[1106] In another embodiment, the inactive Cas9 molecule can be fused to a protein that modifies chromatin. For example, the inactive Cas9 molecule can be fused to heterochromatin protein 1 (HP1), histone lysine methyltransferase (e.g., SUV39H1, SUV39H2, G9A, ESET / SETDB 1, Pr-SET7 / 8, SUV4-20H 1, RIZ1), histone lysine demethylase (e.g., LSD1 / BHC1 10, SpLsd1 / Sw, 1 / Saf1 10, Su(var)3-3, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, Rph 1, JARID 1A / RBP2, JARID1B / PLU-1, JARID 1C / SMCX, JARID1 D / SMCY, Lid, Jhn2, Jmj2), histone lysine deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hos 1, Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hdal, Cir3, SIRT 1, SIRT2, Sir2, Hst 1, Hst2, Hst3, Hst4, HDAC 1 1), and DNA methylase (DNMT1, DNMT2a / DMNT3b / MET1). Inactive Cas9-chromatin modifying molecule fusion proteins can be used to alter chromatin state to reduce target gene expression.

[1107] The heterologous sequence (e.g., a transcriptional repressor domain) can be fused to the N-terminus or C-terminus of the inactive Cas9 protein. In an alternative embodiment, the heterologous sequence (e.g., a transcriptional repressor domain) can be fused to an internal portion (i.e., a portion other than the N-terminus or C-terminus) of the inactive Cas9 protein.

[1108] The ability of the Cas9 molecule / gRNA molecule complex to bind and cut the target nucleic acid can be assessed, for example, by the methods described in Section III herein. The activity of Cas9 molecules (e.g., active Cas9 or inactive Cas9, alone or in a complex with a gRNA molecule) can also be assessed by methods well known in the art, including gene expression assays and chromatin-based assays, such as chromatin immunoprecipitation (ChiP) and chromatin in vivo assays (CiA).

[1109] Other Cas9 fusion molecules

[1110] In an embodiment, the Cas9 molecule (e.g., Cas9 of Streptococcus pyogenes) may further comprise one or more amino acid sequences that confer additional activities.

[1111] In some aspects, the Cas9 molecule can include one or more nuclear localization sequences (NLS), such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In some embodiments, the Cas9 molecule is included at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the amino terminus, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the carboxyl terminus, or a combination of these (e.g., one or more NLS at the amino terminus and one or more NLS at the carboxyl terminus). When there is more than one NLS, each can be selected to be independent of the other NLSs, so that a single NLS can be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered to be near the N-terminus or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain from the N-terminus or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysine or arginine amino acids exposed on the surface of the protein, but other types of NLS are known.Non-limiting examples of NLSs include NLS sequences comprising or derived from: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 206); an NLS from a nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 207)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 208) or RQRRNELKRSP (SEQ ID NO: 209); an hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 210); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 211) from the IBB domain of importin-α. NO:211); the sequence of myoma T protein VSRKRPRP (SEQ ID NO:212) and PPKKARED (SEQ ID NO:213); the sequence of human p53 PQPKKKPL (SEQ ID NO:214); the sequence of mouse c-ab1 IV SALIKKKKKMAP (SEQ ID NO:215); the sequences of influenza virus NS1 DRLRR (SEQ ID NO:216) and PKQKKRK (SEQ ID NO:217); the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO:218); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO:219); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:220); and the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO:221). NO: 221). Other suitable NLS sequences are known in the art (eg, Sorokin, Biochemistry (Moscow) (2007) 72: 13, 1439-1457; Lange J Biol Chem. (2007) 282: 8, 5101-5).

[1112] In one embodiment, the Cas9 molecule (such as Streptococcus pyogenes Cas9 molecule) includes the NLS sequence of SV40, such as arranged at the N-terminus of the Cas9 molecule.In one embodiment, the Cas9 molecule (such as Streptococcus pyogenes Cas9 molecule) includes the NLS sequence of SV40 arranged at the N-terminus of the Cas9 molecule and the NLS sequence of SV40 arranged at the C-terminus of the Cas9 molecule.In one embodiment, the Cas9 molecule (such as Streptococcus pyogenes Cas9 molecule) includes the NLS sequence of SV40 arranged at the N-terminus of the Cas9 molecule and the NLS sequence of the nucleoplasmic protein arranged at the C-terminus of the Cas9 molecule.In any of the above embodiments, the molecule can additionally include a tag, such as a His tag, such as a His (6) tag (SEQ ID NO:247) or a His (8) tag (SEQ ID NO:248), such as at the N-terminus or C-terminus.

[1113] In some aspects, the Cas9 molecule can include one or more amino acid sequences that allow specific identification of the Cas9 molecule, such as tags. In one embodiment, the tag is a histidine tag, such as a histidine tag comprising at least 3, 4, 5, 6, 7, 8, 9, 10 or more histidine amino acids. In an embodiment, the histidine tag is a His6 tag (six histidines) (SEQ ID NO: 247). In other embodiments, the histidine tag is a His8 tag (eight histidines) (SEQ ID NO: 248). In an embodiment, the histidine tag can be separated from one or more other parts of the Cas9 molecule by a joint. In an embodiment, the joint is GGS. An example of this fusion is the Cas9 molecule iProt106520.

[1114] In some aspects, the Cas9 molecule can comprise one or more amino acid sequences recognized by a protease (e.g., comprising a protease cleavage site). In an embodiment, the cleavage site is a tobacco etch virus (TEV) cleavage site, for example, comprising the sequence ENLYFQG (SEQ ID NO: 230). In some aspects, the protease cleavage site (e.g., a TEV cleavage site) is disposed between a tag (e.g., a His tag, such as a His6 (SEQ ID NO: 247) or a His8 tag (SEQ ID NO: 248)) and the rest of the Cas9 molecule. Without being bound by theory, it is believed that this introduction will allow the use of tags for, for example, purification of Cas9 molecules, followed by cleavage, so that the tags do not interfere with Cas9 molecular function.

[1115] In an embodiment, a Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal NLS and a C-terminal NLS (e.g., comprising NLS-Cas9-NLS from N-terminus to C-terminus), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In an embodiment, a Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal NLS, a C-terminal NLS, and a C-terminal His6 tag (SEQ ID NO: 247) (e.g., comprising NLS-Cas9-NLS-His tag from N-terminus to C-terminus), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In an embodiment, a Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal His tag (e.g., a His6 tag (SEQ ID NO: 247)), an N-terminal NLS, and a C-terminal NLS (e.g., comprising His-tag-NLS-Cas9-NLS from N-terminus to C-terminus), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In an embodiment, a Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises a C-terminal NLS and an N-terminal His tag (e.g., a His6 tag (SEQ ID NO: 247)) (e.g., comprising His-tag-Cas9-NLS from N-terminus to C-terminus), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In an embodiment, a Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal NLS and a C-terminal His tag (e.g., a His6 tag (SEQ ID NO: 247)) (e.g., comprising an NLS-Cas9-His tag from N-terminus to C-terminus), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In an embodiment, a Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal His tag (e.g., a His8 tag (SEQ ID NO: 248)), an N-terminal cleavage domain (e.g., a tobacco etch virus (TEV) cleavage domain (e.g., comprising the sequence ENLYFQG (SEQ ID NO: 230))), an N-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., comprising a His tag-TEV-NLS-Cas9-NLS from N-terminus to C-terminus). In any of the above embodiments, Cas9 has the sequence of SEQ ID NO: 205.Alternatively, in any of the above embodiments, the Cas9 has the sequence of a Cas9 variant of SEQ ID NO: 205, e.g., as described herein. In any of the above embodiments, the Cas9 molecule comprises a linker between the His tag and another portion of the molecule, e.g., a GGS linker. The amino acid sequences of the exemplary Cas9 molecules described above are provided below. The "iProt" identifier is the same as Figure 6 The identifier in .

[1116] iProt105026 (also known as iProt106154, iProt106331, iProt106545, and PID426303, depending on how the protein was prepared) (SEQ ID NO: 233):

[1117]

[1118]

[1119] iProt106518 (SEQ ID NO: 234):

[1120]

[1121]

[1122] iProt106519 (SEQ ID NO: 235):

[1123]

[1124] iProt106520 (SEQ ID NO: 236):

[1125]

[1126]

[1127] iProt106521 (SEQ ID NO: 237):

[1128]

[1129]

[1130] iProt106522 (SEQ ID NO: 238):

[1131]

[1132] iProt106658 (SEQ ID NO: 239):

[1133]

[1134]

[1135] iProt106745 (SEQ ID NO: 240):

[1136]

[1137]

[1138] iProt106746 (SEQ ID NO: 241):

[1139]

[1140] iProt106747 (SEQ ID NO: 242):

[1141]

[1142]

[1143] iProt106884 (SEQ ID NO: 243):

[1144]

[1145] iProt 20109496 (SEQ ID NO: 244)

[1146]

[1147] Nucleic acid encoding Cas9 molecule

[1148] Provided herein are nucleic acids encoding Cas9 molecules (e.g., active Cas9 molecules or inactive Cas9 molecules).

[1149] Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al., SCIENCE 2013, 399(6121):819-823; Wang et al., CELL 2013, 153(4):910-918; Mali et al., SCIENCE 2013, 399(6121):823-826; Jinek et al., SCIENCE 2012, 337(6096):816-821.

[1150] In one embodiment, the nucleic acid encoding the Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, for example as described in Section XIII. In one embodiment, the Cas9 mRNA has one or more (e.g., all) of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and / or pseudouridine.

[1151] Additionally or alternatively, the synthetic nucleic acid sequence can be codon-optimized, e.g., at least one uncommon codon or less common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., as described herein.

[1152] Provided below are exemplary codon-optimized nucleic acid sequences encoding the Cas9 molecule of S. pyogenes.

[1153]

[1154]

[1155]

[1156] An exemplary codon-optimized nucleic acid sequence encoding a Cas9 molecule comprising SEQ ID NO: 244 is provided below:

[1157]

[1158]

[1159]

[1160] If the above Cas9 sequence is fused to a peptide or polypeptide at the C-terminus (e.g., an inactive Cas9 is fused to a transcriptional repressor at the C-terminus), it is understood that the stop codon will be removed.

[1161] Also provided herein are nucleic acids / vectors and cells for producing Cas9 molecules, such as Cas9 molecules as described herein. The recombinant production of polypeptide molecules can be completed using technology known to technicians. This paper describes molecules and methods for recombinant production of polypeptide molecules (such as Cas9 molecules, for example, as described herein). As described herein, "recombinant" molecules and production include all polypeptides (such as, for example, Cas9 molecules as described herein) prepared, expressed, produced or separated by a recombinant method, such as polypeptides separated from animals (such as mice) that are transgenic or transchromosomal for nucleic acids encoding target molecules, hybridomas prepared therefrom, molecules such as isolated from transfectomas from host cells transformed to express molecules, molecules separated from recombinant combinatorial libraries, and molecules prepared, expressed, produced or separated by any other method involving all or part of the genes encoding molecules (or parts thereof) being spliced ​​to other DNA sequences. Recombinant production can be from a host cell, for example, a host cell comprising nucleic acids encoding molecules described herein (such as Cas9 molecules, such as Cas9 molecules as described herein).

[1162] Provided herein are nucleic acid molecules encoding molecules (e.g., Cas9 molecules and / or gRNA molecules), e.g., as described herein. Specifically provided are nucleic acid molecules comprising a sequence encoding any one of SEQ ID NO: 233 to SEQ ID NO: 244, or encoding a fragment of any one of SEQ ID NO: 233 to SEQ ID NO: 244, or encoding a polypeptide having at least 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 at least 99% sequence homology to any one of SEQ ID NO: 233 to SEQ ID NO: 244.

[1163] Provided herein are vectors comprising any of the above nucleic acid molecules, eg, vectors as described herein. In embodiments, the nucleic acid molecule is operably linked to a promoter, eg, a promoter operable in a host cell into which the vector is introduced.

[1164] Provided herein are host cells comprising one or more nucleic acid molecules and / or vectors described herein. In an embodiment, the host cell is a prokaryotic host cell. In an embodiment, the host cell is a eukaryotic host cell. In an embodiment, the host cell is a yeast or E. coli cell. In an embodiment, the host cell is a mammalian cell, such as a human cell. Such host cells can be used to produce recombinant molecules described herein, such as Cas9 or gRNA molecules (e.g., as described herein).

[1165] VI. Functional Analysis of Candidate Molecules

[1166] Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecules / gRNA molecule complexes can be evaluated by methods known in the art or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecules are described in, for example, Jinek et al., Science [Science] 2012; 337 (6096): 8 16-821.

[1167] VII. Template Nucleic Acid (for Introducing Nucleic Acid)

[1168] As used herein, the term "template nucleic acid" or "donor template" refers to a nucleic acid to be inserted at or near a target sequence that has been modified (e.g., cut) by the CRISPR system of the present invention. In one embodiment, the nucleic acid sequence at or near the target sequence is modified to have some or all of the sequence of the template nucleic acid, typically at or near one or more cleavage sites. In one embodiment, the template nucleic acid is single-stranded. In an alternative embodiment, the template nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, such as double-stranded DNA. In an alternative embodiment, the template nucleic acid is single-stranded DNA.

[1169] In embodiments, the template nucleic acid comprises a sequence encoding a globin (e.g., a beta globin), e.g., comprises a beta globin gene. In one embodiment, the beta globin encoded by the nucleic acid comprises one or more mutations, e.g., an anti-sickling mutation. In one embodiment, the beta globin encoded by the nucleic acid comprises the mutation T87Q. In one embodiment, the beta globin encoded by the nucleic acid comprises the mutation G16D. In one embodiment, the beta globin encoded by the nucleic acid comprises the mutation E22A. In one embodiment, the beta globin gene comprises the mutations G16D, E22A, and T87Q. In embodiments, the template nucleic acid further comprises one or more regulatory elements, e.g., a promoter (e.g., a human beta globin promoter), a 3' enhancer, and / or at least a portion of a globin locus control region (e.g., one or more DNase I hypersensitivity sites (e.g., HS2, HS3, and / or HS4 of the human beta globin locus)).

[1170] In other embodiments, the template nucleic acid comprises a sequence encoding a gamma globulin, e.g., a gamma globulin gene. In embodiments, the template nucleic acid comprises a sequence encoding more than one copy of a gamma globulin, e.g., comprising two or more (e.g., two) gamma globulin gene sequences. In embodiments, the template nucleic acid further comprises one or more regulatory elements, e.g., a promoter and / or enhancer.

[1171] In one embodiment, the template nucleic acid changes the structure of the target position by participating in a homology-directed repair event. In one embodiment, the template nucleic acid changes the sequence of the target position. In one embodiment, the template nucleic acid causes modified or non-natural bases to be incorporated into the target nucleic acid.

[1172] One of the methods discussed herein can be used to correct mutations in genes or pathways as described herein. In one embodiment, template nucleic acid is used to correct mutations in genes or pathways as described herein by homology directed repair (HDR). In one embodiment, template nucleic acid is used to correct mutations in genes or pathways as described herein by homologous recombination (HR). In one embodiment, template nucleic acid is used to correct mutations in genes or pathways as described herein by non-homologous end joining (NHEJ) repair. In other embodiments, nucleic acids encoding target molecules can be inserted at or near the site modified by the CRISPR system of the present invention. In an embodiment, template nucleic acid comprises regulatory elements, such as one or more promoters and / or enhancers, which are operably linked to the nucleic acid sequence encoding the target molecule, for example, as described herein.

[1173] HDR or HR repair and template nucleic acid

[1174] As described herein, nuclease-induced homology directed repair (HDR) or homologous recombination (HR) can be used to change the target sequence and correct (for example, repair or edit) the mutation in the genome. Although it is not desired to be bound by theory, it is believed that the change of the target sequence occurs by the repair based on the donor template or template nucleic acid. For example, the donor template or template nucleic acid provides the change of the target sequence. It is expected that a plasmid donor or a linear double-stranded template can be used as a template for homologous recombination. It is further expected that a single-stranded donor template can be used as an alternative method (for example, single-strand annealing) for changing the target sequence by the homology directed repair between the target sequence and the donor template. The change to the target sequence achieved by the donor template can depend on the cutting via the Cas9 molecule. The cutting via Cas9 can include double-strand breaks, a single-strand break or two single-strand breaks.

[1175] In one embodiment, the mutation can be corrected by a single double-strand break or two single-strand breaks. In one embodiment, the mutation can be corrected by providing a template and a CRISPR / Cas9 system that generates (1) one double-strand break, (2) two single-strand breaks, (3) two double-strand breaks, wherein the breaks occur on each side of the target sequence, (4) one double-strand break and two single-strand breaks, wherein the double-strand break and two single-strand breaks occur on each side of the target sequence, (5) four single-strand breaks, wherein a pair of single-strand breaks occur on each side of the target sequence, or (6) one single-strand break.

[1176] Double-strand break-mediated correction

[1177] In one embodiment, double-stranded cleavage is affected by a Cas9 molecule having a cleavage activity associated with an HNH-like domain and a cleavage activity associated with a RuvC-like domain (e.g., an N-terminal RuvC-like domain), such as wild-type Cas9. Such embodiments require only a single gRNA.

[1178] Single-strand break-mediated correction

[1179] In other embodiments, two single-strand breaks or gaps are affected by a Cas9 molecule having nickase activity, such as a cleavage activity associated with an HNH-like domain or a cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments require two gRNAs, one for placing each single-strand break. In one embodiment, the Cas9 molecule with nickase activity cuts the chain hybridized to the gRNA, but does not cut the chain complementary to the chain hybridized to the gRNA. In one embodiment, the Cas9 molecule with nickase activity does not cut the chain hybridized to the gRNA, but cuts the chain complementary to the chain hybridized to the gRNA.

[1180] In one embodiment, the nickase has HNH activity, for example, to inactivate a Cas9 molecule having RuvC activity (e.g., a Cas9 molecule having a mutation at D10 (e.g., a D10A mutation). D10A inactivates RuvC; thus, the Cas9 nickase (only) has HNH activity and will cleave the strand hybridized to the gRNA (e.g., the complementary strand, which does not have an NGG PAM on it). In other embodiments, a Cas9 molecule having a mutation at H840 (e.g., H840A) can be used as a nickase. H840A inactivates HNH; thus, the Cas9 nickase (only) has RuvC activity and cleaves the non-complementary strand (e.g., a strand having an NGG PAM and the same sequence as the gRNA).

[1181] In one embodiment of nickase and two gRNAs for locating two single-stranded nicks, one nick is located on the + strand of the target nucleic acid, and one nick is located on the - strand. The PAM faces outward. gRNA can be selected so that the gRNAs are separated by about 0-50, 0-100, or 0-200 nucleotides. In one embodiment, there is no overlap between the target sequences complementary to the targeting domains of the two gRNAs. In one embodiment, the gRNAs do not overlap and are separated by up to 50, 100, or 200 nucleotides. In one embodiment, using two gRNAs can increase specificity, for example, by reducing off-target binding (Ran et al., CELL [cell] 2013).

[1182] In one embodiment, a single nick can be used to induce HDR. It is contemplated herein that a single nick can be used to increase the rate of HDR, HR, or NHEJ at a given cleavage site.

[1183] Placement of double-strand breaks or single-strand breaks relative to target locations

[1184] Double-strand breaks or the single-strand breaks in a chain should be close enough to the target position so that correction occurs. In one embodiment, the distance is no more than 50, 100, 200, 300, 350 or 400 nucleotides. Although not wishing to be bound by theory, it is believed that the fracture should be close enough to the target position so that the fracture is subjected to the removal zone of exonuclease mediation during the end resection process. If the distance between the target position and the fracture is too large, the sudden change may not be included in the end resection, and therefore may not be corrected, because the donor sequence can only be used to correct the sequence in the end resection zone.

[1185] In one embodiment where a gRNA (single molecule (or chimeric) or modular gRNA) and a Cas9 nuclease induce double strand breaks to induce HDR- or HR-mediated correction, the cleavage site is between 0-200 bp from the target position (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp). In one embodiment, the cleavage site is between 0-100 bp from the target position (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75, or 75 to 100 bp).

[1186] In one embodiment where two gRNAs (independently, unimolecular (or chimeric) or modular gRNAs) complexed with the Cas9 nickase induce two single-strand breaks to induce HDR-mediated correction, the closer nick is between 0-200 bp from the target position (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 50). 5 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp), and the two nicks will ideally be within 25-55 bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp) and no more than 100 bp from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 bp from each other). In one embodiment, the cleavage site is between 0-100 bp from the target position (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75, or 75 to 100 bp).

[1187] In one embodiment, two gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to position double-strand breaks on both sides of the target position. In an alternative embodiment, three gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to position double-strand breaks (i.e., one gRNA complexed with Cas9 nuclease) and two single-strand breaks or paired single-strand breaks (i.e., two gRNA complexed with Cas9 nickase) on either side of the target position (e.g., the first gRNA is used to target the upstream (i.e., 5') of the target position, and the second gRNA is used to target the downstream (i.e., 3') of the target position). In another embodiment, four gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to produce two pairs of single-strand breaks (i.e., two pairs of two gRNA complexed with Cas9 nickase) on either side of the target position (e.g., the first gRNA is used to target the upstream (i.e., 5') of the target position, and the second gRNA is used to target the downstream (i.e., 3') of the target position). The closer of one or more double-strand breaks or two paired single-stranded nicks will ideally be within 0-500bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50, or 25bp from the target position). When using nickase, the two paired nicks are within 25-55bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45bp) and no more than 100bp from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, or 10bp).

[1188] In one embodiment, two gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to position double-strand breaks on both sides of the target position. In an alternative embodiment, three gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to position double-strand breaks (i.e., one gRNA is complexed with the Cas9 nuclease) and two single-strand breaks or paired single-strand breaks (i.e., two gRNAs are complexed with the Cas9 nickase) on two target sequences (e.g., the first gRNA is used to target the upstream (i.e., 5') target sequence of the insertion site, and the second gRNA is used to target the downstream (i.e., 3') target sequence of the insertion site). In another embodiment, four gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to produce two pairs of single-strand breaks (i.e., two pairs of two gRNAs are complexed with the Cas9 nickase) on either side of the insertion site (e.g., the first gRNA is used to target the upstream (i.e., 5') target sequence described herein, and the second gRNA is used to target the downstream (i.e., 3') target sequence described herein). The closer of one or more double-strand breaks or two paired single-stranded nicks will ideally be within 0-500bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50, or 25bp from the target position). When using nickase, the two paired nicks are within 25-55bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45bp) and no more than 100bp from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, or 10bp).

[1189] Length of homology arms

[1190] Homology arms should at least extend to the region where end excision can occur, for example, to allow the single-stranded overhang of excision to search for the complementary region in the donor template. Total length may be limited by parameters such as plasmid size or viral packaging restrictions. In one embodiment, homology arms do not extend into repetitive elements (e.g., ALU repeats, LINE repeats). Templates can have two homology arms of identical or different lengths.

[1191] Exemplary homology arm lengths include at least 25, 50, 100, 250, 500, 750, or 1000 nucleotides.

[1192] As used herein, target position refers to a site on a target nucleic acid (e.g., chromosome) modified by a Cas9 molecule dependent process. For example, the target position can be a target position for modification (e.g., correction) guided by a modified Cas9 molecule cutting and template nucleic acid of a target nucleic acid. In one embodiment, the target position can be a site between two nucleotides (e.g., adjacent nucleotides) on a target nucleic acid to which one or more nucleotides are added. The target position can include one or more nucleotides that are changed (e.g., corrected) by a template nucleic acid. In one embodiment, the target position is within a target sequence (e.g., a sequence bound to a gRNA). In one embodiment, the target position is upstream or downstream of a target sequence (e.g., a sequence bound to a gRNA).

[1193] In one embodiment, the template nucleic acid comprises a sequence corresponding to a site on the target sequence that is cut by a cleavage event mediated by Cas9. In one embodiment, the template nucleic acid comprises a sequence corresponding to a site on the target sequence that is cut by a cleavage event mediated by Cas9. In one embodiment, the template nucleic acid comprises a sequence corresponding to both (a first site on the target sequence that is cut in the first Cas9-mediated event and a second site on the target sequence that is cut in the second Cas9-mediated event).

[1194] In one embodiment, the template nucleic acid may comprise a sequence that causes an alteration in the coding sequence of the translated sequence, such as causing one amino acid in the protein product to be substituted for another amino acid, such as causing a mutant allele to be converted to a wild-type allele, a wild-type allele to be converted to a mutant allele, and / or a sequence that introduces a stop codon, inserts an amino acid residue, deletes an amino acid residue, or a nonsense mutation.

[1195] In other embodiments, the template nucleic acid may comprise sequences that result in changes in non-coding sequences, such as in exons or in 5' or 3' untranslated or non-transcribed regions. Such changes include changes in control elements (e.g., promoters, enhancers), as well as changes in cis- or trans-acting control elements.

[1196] The template nucleic acid may comprise a sequence that, when integrated, results in:

[1197] Reduce the activity of positive control elements;

[1198] Increase the activity of positive control elements;

[1199] Reduce the activity of negative control elements;

[1200] Increase the activity of negative control elements;

[1201] Reduce gene expression;

[1202] Increase gene expression;

[1203] Increased resistance to a condition or disease;

[1204] Increased resistance to viral entry;

[1205] Correct mutations or change unwanted amino acid residues;

[1206] Conferring, increasing, eliminating, or decreasing a biological property of a gene product, such as increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule.

[1207] The template nucleic acid may comprise a sequence that results in:

[1208] The target sequence is altered in the sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides.

[1209] In one embodiment, the length of the template nucleic acid is 20+ / -10, 30+ / -10, 40+ / -10, 50+ / -10, 60+ / -10, 70+ / -10, 80+ / -10, 90+ / -10, 100+ / -10, 110+ / -10, 120+ / -10, 130+ / -10, 140+ / -10, 150+ / -10, 160+ / -10, 170+ / -10, -10, 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, 2000-3000 or more than 3000 nucleotides.

[1210] The template nucleic acid contains the following components:

[1211] [5' homology arm]-[insertion sequence]-[3' homology arm].

[1212] The homology arms provide for recombination into the chromosome, which can replace an undesirable element, such as a mutation or feature, with a replacement sequence. In one embodiment, the homology arms flank the most distal cleavage site.

[1213] In one embodiment, the 3' end of the 5' homology arm is located near the 5' end of the replacement sequence. In one embodiment, the 5' homology arm can extend at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides 5' from the 5' end of the replacement sequence.

[1214] In one embodiment, the 5' end of the 3' homology arm is located near the 3' end of the replacement sequence. In one embodiment, the 3' homology arm can extend at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 nucleotides 3' from the 3' end of the replacement sequence.

[1215] It is contemplated herein that one or both homology arms can be shortened to avoid inclusion of certain sequence repeat elements, such as Alu repeats, LINE elements. For example, the 5' homology arm can be shortened to avoid inclusion of a sequence repeat element. In other embodiments, the 3' homology arm can be shortened to avoid inclusion of a sequence repeat element. In certain embodiments, both the 5' homology arm and the 3' homology arm can be shortened to avoid inclusion of a certain sequence repeat element.

[1216] It is contemplated herein that the template nucleic acid for correction of mutations can be designed to be used as single-stranded oligonucleotides (ssODNs). When using ssODNs, the length of the 5' homology arm and the 3' homology arm can be in the range of up to about 200 base pairs (bp), for example, at least 25, 50, 75, 100, 125, 150, 175 or 200 bp in length. Longer homology arms are also contemplated for ssODNs because improvements in oligonucleotide synthesis continue.

[1217] NHEJ methods for gene targeting

[1218] As described herein, nuclease-induced non-homologous end joining (NHEJ) can be used for target gene specific knockout. Nuclease-induced NHEJ can also be used to remove (e.g., delete) sequences in the gene of interest.

[1219] Although not wishing to be bound by theory, it is believed that, in one embodiment, the genomic alterations associated with the methods described herein rely on the error-prone nature of nuclease-induced NHEJ and NHEJ repair pathways. NHEJ repairs double-strand breaks in DNA by connecting two ends together; however, generally, as long as two compatible ends are perfectly connected just when they are formed by double bond breakage, the original sequence is restored. Before the ends are reconnected, the DNA ends of double bond breakage are often the subjects of enzyme processing, which produces the addition or removal of nucleotides at one or both chains. This allows the DNA sequence at the NHEJ repair site to have insertion and / or deletion (indel) mutations. Two-thirds of these mutations can change the reading frame and therefore produce non-functional proteins. In addition, mutations that maintain the reading frame but insert or delete a large amount of sequences can destroy the function of the protein. This is locus dependent because mutations in key functional domains may be less tolerant to mutations than those in the non-critical regions of the protein.

[1220] The insertion / deletion mutations generated by NHEJ are unpredictable in nature; however, at a given break site, certain insertion / deletion sequences are favored and over-represented in groups. The length of the deletion can vary widely; the most common are in the range of 1-50 bp, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often contain short repeats of the sequence closely surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence is often traced to other regions of the genome or to plasmid DNA present in the cell.

[1221] Because NHEJ is a method of mutagenesis, it can also be used to delete small sequence motifs, as long as there is no need to produce a specific final sequence. If double-strand breaks are targeted near a short target sequence, the deletion mutation caused by NHEJ repair often spans and therefore removes unwanted nucleotides. For the disappearance of larger DNA segments, introducing two double-strand breaks (one double-strand break on each side of the sequence) can produce NHEJ between the ends, wherein the entire intervening sequence is removed. Both methods can be used to delete specific DNA sequences; However, the fallibility of NHEJ may still produce insertion / deletion mutations at the repair site.

[1222] Double-stranded cutting Cas9 molecules and single-stranded or nickase Cas9 molecules can be used in methods and compositions described herein to produce the insertion / deletion mediated by NHEJ. The insertion / deletion mediated by NHEJ of a targeted gene (such as a coding region, such as an early coding region of a gene of interest) can be used to knock out the gene of interest (i.e., eliminate its expression). For example, the early coding region of a gene of interest includes the sequence immediately following the transcription start site, in the first exon of the coding sequence, or in 500bp of the transcription start site (for example, less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50bp).

[1223] Placement of double-strand or single-strand breaks relative to target locations

[1224] In one embodiment where gRNA and Cas9 nuclease generate double-strand breaks to induce NHEJ-mediated insertions / deletions, gRNA (e.g., single (or chimeric) or modular gRNA molecules) are configured to position a double-strand break immediately adjacent to the nucleotides at the target position. In one embodiment, the cleavage site is between 0-500 bp from the target position (e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 bp from the target position).

[1225] In one embodiment where two gRNAs complexed with the Cas9 nickase induce two single-strand breaks to induce NHEJ-mediated insertions / deletions, the two gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to locate the two single-strand breaks to provide nucleotides at the target location for NHEJ repair. In one embodiment, the gRNAs are configured to locate the nicks at the same location on different strands or within a few nucleotides of each other, essentially mimicking a double-strand break. In one embodiment, the closer nick is between 0-30bp from the target position (e.g., less than 30, 25, 20, 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1bp from the target position), and the two nicks are within 25-55bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45bp) and no more than 100bp from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, or 10bp). In one embodiment, the gRNA is configured to place a single-strand break on either side of the nucleotide at the target position.

[1226] Double-stranded cutting Cas9 molecules and single-stranded or nickase Cas9 molecules can be used in the methods and compositions described herein to produce the fractures on both sides of the target position. Double-stranded breaks or paired single-stranded breaks can be produced on both sides of the target position to remove the nucleic acid sequence between the two nicks (for example, to delete the region between the two breaks). In one embodiment, two gRNAs (for example, independently, single molecules (or chimeric) or modular gRNAs) are configured to position double-stranded breaks on both sides of the target position (for example, the first gRNA is used to target the upstream (i.e. 5') of the mutation in the gene or approach as described herein, and the second gRNA is used to target the downstream (i.e. 3') of the mutation in the gene or approach as described herein). In an alternative embodiment, three gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to position double-strand breaks (i.e., one gRNA complexed with the Cas9 nuclease) and two single-strand breaks or pairs of single-strand breaks (i.e., two gRNAs complexed with the Cas9 nickase) on either side of the target position (e.g., a first gRNA is used to target upstream (i.e., 5') of a mutation in a gene or pathway described herein, and a second gRNA is used to target downstream (i.e., 3') of a mutation in a gene or pathway described herein). In another embodiment, four gRNAs (e.g., independently, unimolecular (or chimeric) or modular gRNAs) are configured to generate two pairs of single-strand breaks (i.e., two pairs of two gRNAs complexed with the Cas9 nickase) on either side of the target position (e.g., a first gRNA is used to target upstream (i.e., 5') of a mutation in a gene or pathway described herein, and a second gRNA is used to target downstream (i.e., 3') of a mutation in a gene or pathway described herein). The closer of one or more double-strand breaks or two paired single-stranded nicks will ideally be within 0-500bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50, or 25bp from the target position). When using nickase, the two paired nicks are within 25-55bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45bp) and no more than 100bp from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, or 10bp).

[1227] In other embodiments, insertion of the template nucleic acid can be mediated by microhomology end joining (MMEJ). See, for example, Saksuma et al., "MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems." Nature Protocols 11, 118-133 (2016) doi: 10.1038 / nprot.2015.140, published online December 17, 2015, which is incorporated by reference in its entirety.

[1228] VIII. Systems Comprising More Than One gRNA Molecule

[1229] Although not intended to be bound by theory, it has been surprisingly shown herein that two target sequences of targeting adjacent continuous nucleic acids (for example, by two gRNA molecules / Cas9 molecule complexes, each of which induces single-strand or double-strand breaks at or near its corresponding target sequence) induce the excision (for example, deletion) of the nucleic acid sequence (or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% nucleic acid sequence) between the two target sequences. In some aspects, present disclosure provides the purposes of two or more gRNA molecules, which include targeting domains targeting target sequences, which are adjacent to continuous nucleic acids, and these continuous nucleic acids are, for example, chromosomes, such as genes or loci, including introns, exons, and regulatory elements. This purpose can be achieved, for example, by introducing two or more gRNA molecules into cells together with one or more Cas9 molecules (or nucleic acids encoding two or more gRNA molecules and / or one or more Cas9 molecules).

[1230] In some aspects, the target sequences of two or more gRNA molecules are positioned to be separated by at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000 or 15,000 nucleotides on continuous nucleic acid, but no more than 25,000 nucleotides apart on continuous nucleic acid. In one embodiment, the target sequence is positioned to be separated by about 4000 to about 6000 nucleotides. In one embodiment, the target sequence is positioned to be separated by about 4000 nucleotides. In one embodiment, the target sequences are located about 5000 ...

Claims

1. A gRNA molecule comprising tracr and crRNA, wherein the crRNA comprises a targeting domain, wherein the targeting domain: a) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,250,094-5,250,237, -strand, hg38; b) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,255,022-5,255,164, -strand, hg38; c) complementary to a target sequence of a non-deleted HFPH region (e.g., a non-deleted human HPFH region); d) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,249,833 to Chr11:5,250,237, -strand, hg38; e) complementary to a target sequence within a genomic nucleic acid sequence at Chr11:5,254,738 to Chr11:5,255,164, first strand, hg38; f) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,249,833-5,249,927, -strand, hg38; g) complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,254,738-5,254,851, -strand, hg38; h) is complementary to a target sequence within the genomic nucleic acid sequence at Chr11:5,250,139-5,250,237, first strand, hg38; or i) combinations thereof.

2. The gRNA molecule of claim 1, wherein the targeting domain comprises, for example, consists of, any one of: SEQ ID NO: 1 to SEQ ID NO: 72 or fragments thereof.

3. The gRNA molecule of claim 2, wherein the targeting domain comprises, for example, consists of, any one of SEQ ID NO:67, 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:28, SEQ ID NO:34, SEQ ID 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.

4. The gRNA molecule of claim 2, wherein the targeting domain comprises, for example, consists of, any one of: a) SEQ ID NO:67, 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 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 a fragment thereof.

5. The gRNA molecule of any one of claims 2 to 4, wherein the targeting domain comprises, for example, consists of, 17, 18, 19 or 20 consecutive nucleic acids of any one of the listed targeting domain sequences.

6. The gRNA molecule of claim 5, wherein any one of the 17, 18, 19, or 20 consecutive nucleic acids of the listed targeting domain sequence is 17, 18, 19, or 20 consecutive nucleic acids arranged at the 3' end of the listed targeting domain sequence.

7. The gRNA molecule of claim 5, wherein any one of the 17, 18, 19, or 20 consecutive nucleic acids of the listed targeting domain sequence is 17, 18, 19, or 20 consecutive nucleic acids arranged at the 5' end of the listed targeting domain sequence.

8. The gRNA molecule of claim 5, wherein any 17, 18, 19, or 20 consecutive nucleic acids of the listed targeting domain sequence do not comprise the 5' or 3' nucleic acid of the listed targeting domain sequence.

9. The gRNA molecule of any one of claims 2-8, wherein the targeting domain consists of the enumerated targeting domain sequence.

10. The gRNA molecule of any one of claims 1-9, wherein the targeting domain comprises, e.g., consists of, SEQ ID NO: 67.

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