COMPOSITIONS AND METHODS FOR THE TREATMENT OF HEMOGLOBINOPATHIES

BACKGROUND

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria as an adaptive immune system to defend against viral attack. Upon exposure to a virus, 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 that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.

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

SUMMARY OF THE INVENTION

Without being bound by theory, the invention is based in part on the discovery that CRISPR systems, e.g., Cas9 CRISPR systems, e.g., as described herein, can be used to modify cells (e.g., hematopoietic stem and progenitor cells (HSPCs)) to increase fetal hemoglobin (HbF) expression and/or decrease expression of beta globin (e.g., a beta globin gene having a disease-causing mutation), and that such cells may be used to treat hemoglobinopathies, e.g., sickle cell disease and beta thalassemia.

Thus, in an aspect, the invention provides CRISPR systems (e.g., Cas CRISPR systems, e.g., Cas9 CRISPR systems, e.g., S. pyogenes Cas9 CRISPR systems) comprising one or more, e.g., one, gRNA molecule as described herein. Any of the gRNA molecules described herein may be used in such systems, and in the methods and cells described herein.

In an aspect, the invention provides a gRNA molecule that includes a tracr and crRNA, wherein the crRNA includes a targeting domain that is complementary with a target sequence of the BCL11A gene, a BCL11a enhancer, or a HFPH region.

In another aspect, the invention provides a gRNA molecule that includes a targeting domain that is complementary with a target sequence of the BCL11A gene, e.g., is complementary with a target sequence within a BCL11a coding region (e.g., within a BCL11a exon, e.g., within BCL11a exon 2). In embodiments, the gRNA comprises a targeting domain that includes, e.g., consists of, any one of SEQ ID NO: 1 to SEQ ID NO: 85 or SEQ ID NO: 400 to SEQ ID NO: 1231.

In another aspect, the invention provides a gRNA molecule that includes a targeting domain that is complementary with a target sequence of a BCL11A enhancer.

In embodiments, the gRNA comprises a targeting domain that includes, e.g., consists of, any one of SEQ ID NO: 1232 to SEQ ID NO: 1499.

In embodiments, the gRNA to a target sequence of a BCL11a enhancer is to a target sequence within the +58 region of the BCL11a enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 182 to SEQ ID NO: 277 or SEQ ID NO: 334 to SEQ ID NO: 341. In embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 341, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 247, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO: 244, SEQ ID NO: 199, SEQ ID NO: 251, SEQ ID NO: 250, SEQ ID NO: 334, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 336, or SEQ ID NO: 337. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 248. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 247. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 245. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 336. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 337. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 338. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 335. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 252. In an embodiment, the gRNA is a dgRNA that includes a crRNA that includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 248-SEQ ID NO: 6607, and a tracr that includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 6660. In an embodiment, the gRNA is a dgRNA that includes a crRNA that includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 247-SEQ ID NO: 6607, and a tracr that includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 6660. In an embodiment, the gRNA molecule is a sgRNA molecule and includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 338-SEQ ID NO: 6604-UUUU. In an embodiment, the gRNA molecule is a sgRNA molecule and includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 335-SEQ ID NO: 6604-UUUU. In an embodiment, the gRNA molecule is a sgRNA molecule and includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 336-SEQ ID NO: 6604-UUUU. In an embodiment, the gRNA molecule is a sgRNA molecule and includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 245-SEQ ID NO: 6604-UUUU. In an embodiment, the gRNA molecule is a sgRNA molecule and includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 337-SEQ ID NO: 6604-UUUU. In an embodiment, the gRNA molecule is a sgRNA molecule and includes, e.g., consists of, e.g., from 5′ to 3′, SEQ ID NO: 252-SEQ ID NO: 6604-UUUU.

In embodiments, the gRNA to a target sequence of a BCL11a enhancer is to a target sequence within the +62 region of the BCL11a enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 278 to SEQ ID NO: 333. In embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 318, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 294, SEQ ID NO: 310, SEQ ID NO: 319, SEQ ID NO: 298, SEQ ID NO: 322, SEQ ID NO: 311, SEQ ID NO: 315, SEQ ID NO: 290, SEQ ID NO: 317, SEQ ID NO: 309, SEQ ID NO: 289, or SEQ ID NO: 281. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 318.

In embodiments, the gRNA to a target sequence of a BCL11a enhancer is to a target sequence within the +55 region of the BCL11a enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 1596 to SEQ ID NO: 1691. In embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 1683, SEQ ID NO: 1638, SEQ ID NO: 1647, SEQ ID NO: 1609, SEQ ID NO: 1621, SEQ ID NO: 1617, SEQ ID NO: 1654, SEQ ID NO: 1631, SEQ ID NO: 1620, SEQ ID NO: 1637, SEQ ID NO: 1612, SEQ ID NO: 1656, SEQ ID NO: 1619, SEQ ID NO: 1675, SEQ ID NO: 1645, SEQ ID NO: 1598, SEQ ID NO: 1599, SEQ ID NO: 1663, SEQ ID NO: 1677, or SEQ ID NO: 1626.

In another aspect, the invention provides a gRNA molecule that includes a targeting domain that is complementary with a target sequence of a hereditary persistence of fetal hemoglobin (HPFH) region. In an embodiment, the HPFH region is the French HPFH region. In embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 86 to SEQ ID NO: 181 or SEQ ID NO: 1500 to SEQ ID NO: 1595. In embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 100, SEQ ID NO: 165, SEQ ID NO: 113, SEQ ID NO: 99, SEQ ID NO: 112, SEQ ID NO: 98, SEQ ID NO: 1580, SEQ ID NO: 106, SEQ ID NO: 1503, SEQ ID NO: 1589, SEQ ID NO: 160, SEQ ID NO: 1537, SEQ ID NO: 159, SEQ ID NO: 101, SEQ ID NO: 162, SEQ ID NO: 104, SEQ ID NO: 138, SEQ ID NO: 1536, SEQ ID NO: 1539, SEQ ID NO: 1585. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 100. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 165. In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 113.

In any of the aforementioned embodiments, the gRNA molecule may further have regions and/or properties described herein. In an aspect, the gRNA molecule (e.g., of any of the aforementioned aspects or embodiments) includes a targeting domain that includes, e.g., consists of, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleic acids of any one of the recited targeting domain sequences. In embodiments, the 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleic acids of any one of the recited targeting domain sequences are the 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleic acids disposed at the 3′ end of the recited targeting domain sequence. In embodiments, the 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleic acids of any one of the recited targeting domain sequences are the 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleic acids disposed at the 5′ end of the recited targeting domain sequence. In embodiments, the 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleic acids of any one of the recited targeting domain sequences do not include either the 5′ or 3′ nucleic acid of the recited targeting domain sequence. In any of the aforementioned aspects or embodiments, the targeting domain may consist of the recited targeting domain sequence.

In embodiments of the gRNA molecule, including in any of the aforementioned aspects and embodiments, the targeting domain includes 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids of any one of the recited targeting domain sequences. In other embodiments of the gRNA molecule, including in any of the aforementioned aspects and embodiments, the targeting domain consists of 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), or 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids of any one of the recited targeting domain sequences. In embodiments of the gRNA molecule, including in any of the aforementioned aspects and embodiments, the 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), or 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids of any one of the recited targeting domain sequences are the 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), or 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids disposed at the 3′ end of the recited targeting domain sequence. In other embodiments of the gRNA molecule, including in any of the aforementioned aspects and embodiments, the 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), or 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids of any one of the recited targeting domain sequences are the 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), or 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids disposed at the 5′ end of the recited targeting domain sequence. In other embodiments of the gRNA molecule, including in any of the aforementioned aspects and embodiments, the 17, 18, 19, 20, 21 (if present in the reference sequence), 22 (if present in the reference sequence), 23 (if present in the reference sequence), or 24 (if present in the reference sequence), or 25 (if present in the reference sequence) consecutive nucleic acids of any one of the recited targeting domain sequences do not include either the 5′ or 3′ nucleic acid of the recited targeting domain sequence.

In an aspect, including in any of the aforementioned aspects or embodiments, the gRNA molecule includes a portion of the crRNA and a portion of the tracr that hybridize to form a flagpole, that includes SEQ ID NO: 6584 or 6585. In embodiments, the flagpole further includes a first flagpole extension, located 3′ to the crRNA portion of the flagpole, wherein said first flagpole extension includes SEQ ID NO: 6586. In embodiments, the flagpole further includes a second flagpole extension located 3′ to the crRNA portion of the flagpole and, if present, the first flagpole extension, wherein said second flagpole extension includes SEQ ID NO: 6587.

In an aspect, including in any of the aforementioned aspects or embodiments, the invention provides a gRNA molecule that includes a tracr that includes, e.g., consists of, SEQ ID NO: 6660 or SEQ ID NO: 6661. In embodiments, the crRNA portion of the flagpole includes SEQ ID NO: 6607 or SEQ ID NO: 6608.

In an aspect, including in any of the aforementioned aspects or embodiments, the invention provides a gRNA molecule that includes a tracr that includes SEQ ID NO: 6589 or 6590, and optionally, if a first flagpole extension is present, a first tracr extension, disposed 5′ to SEQ ID NO: 6589 or 6590, said first tracr extension including SEQ ID NO: 6591.

In an aspect, including in any of the aforementioned aspects or embodiments, the invention provides a gRNA molecule wherein the targeting domain and the tracr are disposed on separate nucleic acid molecules (e.g., a dgRNA molecule).

In an aspect, including in any of the aforementioned aspects or embodiments, the invention provides a gRNA molecule wherein the targeting domain and the tracr are disposed on a single nucleic acid molecule (e.g., a sgRNA molecule), and wherein the tracr is disposed 3′ to the targeting domain. In embodiments, the sgRNA molecule includes a loop, disposed 3′ to the targeting domain and 5′ to the tracr, e.g., a loop including, e.g., consisting of, SEQ ID NO: 6588.

In an aspect, including in any of the aforementioned aspects or embodiments, the invention provides a gRNA molecule including, from 5′ to 3′, [targeting domain]-:

(a) SEQ ID NO: 6601;
(b) SEQ ID NO: 6602;
(c) SEQ ID NO: 6603;
(d) SEQ ID NO: 6604; or

(e) any of (a) to (d), above, further including, at the 3′ end, 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g., 4 uracil nucleotides. In embodiments, there are no intervening nucleotides between the targeting domain and the sequence of any of (a)-(e).

In another aspect, the invention provides CRISPR systems, e.g., Cas CRISPR systems, e.g., Cas9 CRISPR systems, e.g., S. pyogenes Cas9 CRISPR systems, the include one or more, e.g., one, gRNA molecule of any of the aforementioned aspects and embodiments. In an aspect, the invention provides a composition including a first gRNA molecule of any of the aforementioned aspects and embodiments, further including a Cas9 molecule. In embodiments, the Cas9 molecule is an active or inactive s. pyogenes Cas9. In embodiments, the first gRNA molecule and Cas9 molecule are present in a ribonuclear protein complex (RNP).

In another aspect, the invention provides compositions that include more than one gRNA, e.g., more than one gRNA molecule as described herein, e.g., more than one gRNA molecule of any of the aforementioned gRNA molecule aspects or embodiments. Thus, in a further aspect, the invention provides a composition of any of the aforementioned composition aspects and embodiments, further including a second gRNA molecule; a second gRNA molecule and a third gRNA molecule; or a second gRNA molecule, a third gRNA molecule, and a fourth gRNA molecule, wherein the second gRNA molecule, the third gRNA molecule (if present), and the fourth gRNA molecule (if present) are a gRNA molecule as described herein, e.g., a gRNA molecule of any of the aforementioned gRNA molecule aspects or embodiments. In an embodiment, each gRNA molecule of the composition is complementary to a different target sequence. In an embodiment, each gRNA molecule is complementary to target sequences within the same gene or region. In an aspect, the first gRNA molecule, the second gRNA molecule, the third gRNA molecule (if present), and the fourth gRNA molecule (if present) are complementary to target sequences not more than 20000 nucleotides, not more than 10000 nucleotides, not more than 6000, not more than 5000 nucleotides, not more than 4000, not more than 1000 nucleotides, not more than 500 nucleotides, not more than 400 nucleotides, not more than 300 nucleotides, not more than 200 nucleotides, not more than 100 nucleotides, not more than 90 nucleotides, not more than 80 nucleotides, not more than 70 nucleotides, not more than 60 nucleotides, not more than 50 nucleotides, not more than 40 nucleotides, not more than 30 nucleotides, not more than 20 nucleotides or not more than 10 nucleotides apart. In another aspect, each gRNA molecule of the composition is complementary to target sequence within a different gene or region.

Specific and preferred combinations of more than one gRNA molecule of the invention are described herein. In an aspect, the composition includes a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and second gRNA molecule are complementary to different target sequences, and are:

(a) independently selected from the gRNA molecules described herein (e.g., above) to the BCL11a gene;

(b) independently selected from the gRNA molecules described herein (e.g., above) to the +58 BCL11a enhancer;

(c) independently selected from the gRNA molecules described herein (e.g., above) to the +62 BCL11a enhancer;

(d) independently selected from the gRNA molecules described herein (e.g., above) to the +55 BCL11a enhancer; or

(e) independently selected from the gRNA molecules described herein (e.g., above) to a HPFH region.

In an aspect, the composition includes a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and second gRNA molecule are complementary to different target sequences, and:

(a) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the BCL11a gene, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +58 BCL11a enhancer;

(b) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the BCL11a gene, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +62 BCL11a enhancer;

(c) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the BCL11a gene, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +55 BCL11a enhancer;

(d) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the BCL11a gene, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to a HPFH region;

(e) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +58 BCL11a enhancer, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +62 BCL11a enhancer;

(f) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +58 BCL11a enhancer, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +55 BCL11a enhancer;

(g) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +58 BCL11a enhancer, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to a HPFH region;

(h) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +62 BCL11a enhancer, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +55 BCL11a enhancer;

(i) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +62 BCL11a enhancer, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to a HPFH region;

(j) the first gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to the +55 BCL11a enhancer, and the second gRNA molecule is selected from the gRNA molecules described herein (e.g., above) to a HPFH region;

In another aspect, the composition that includes a first gRNA molecule and a second gRNA molecule, includes:

(a) a first gRNA molecule that is selected from the gRNA molecules described herein (e.g., above) to a HPFH region, and a second gRNA molecule includes a targeting domain that is complementary to a target sequence of the beta globin gene; or

(b) a first gRNA molecule that is selected from the gRNA molecules described herein (e.g., above) to a BCL11a enhancer, e.g., a +58 BCL11a enhancer, a +55 BCL11a enhancer or a +62 BCL11a enhancer, and a second gRNA molecule includes a targeting domain that is complementary to a target sequence of the beta globin gene.

In any of the aforementioned composition aspects and embodiments, the composition may consist of, with respect to the gRNA components of the composition, a first gRNA molecule and a second gRNA molecule.

In any of the aforementioned composition aspects and embodiments, the compositions may be formulated in a medium suitable for electroporation.

In another aspect, the invention provides nucleic acids encoding the gRNA molecule(s) and/or Cas molecules of the CRISPR systems. Without being bound by theory, it is believed that delivering such nucleic acids to cells will lead to the expression of the CRISPR system within the cell. In an aspect, the invention provides a nucleic acid sequence that encodes one or more gRNA molecules of any of the previous gRNA aspects and embodiments. In embodiments, the nucleic acid includes a promoter operably linked to the sequence that encodes the one or more gRNA molecules. In embodiments, the promoter is a promoter recognized by an RNA polymerase II or RNA polymerase III. In embodiments, the promoter is a U6 promoter or an HI promoter.

In further aspects, the nucleic acid further comprises sequence encoding a Cas9 molecule. In embodiments, the nucleic acid includes a promoter operably linked to the sequence that encodes a Cas9 molecule. In embodiments, the promoter is an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, an ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.

In an aspect the invention provides a vector that includes the nucleic acid of any of the previous 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 viral (AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, and an RNA vector.

In another aspect, the invention provides a composition including one or more gRNA molecules, e.g., as described herein, e.g., in any of the previous gRNA molecule aspects and embodiments, and nucleic acid encoding a Cas9 molecule.

In another aspect, the invention provides a composition including nucleic acid encoding one or more gRNA molecules (e.g., as described herein, e.g., of any of the previous gRNA molecule aspects and embodiments), and a Cas9 molecule.

In another aspect, the invention provides a composition, e.g., a composition of any of the aforementioned composition aspects and embodiments, further including a template nucleic acid. In another aspect, the invention provides a composition, e.g., a composition of any of the aforementioned composition aspects and embodiments, further including nucleic acid sequence encoding a template nucleic acid. In embodiments, the template nucleic acid includes a nucleotide that corresponds to a nucleotide of a target sequence of the gRNA molecule. In embodiments, the template nucleic acid includes nucleic acid encoding human beta globin, e.g., human beta globin including one or more of the mutations G16D, E22A and T87Q. In embodiments, the template nucleic acid includes nucleic acid encoding human gamma globin.

In another aspect, the invention provides cells that include (or at any time included) a gRNA molecule, CRISPR system, composition or nucleic acid (e.g., as described herein, e.g., as in any of the aforementioned aspects and embodiments). In such aspects, the invention provides cells that have been modified by such inclusion.

Thus, in an aspect, the invention provides a method of altering e.g., altering the structure, e.g., sequence, of a target sequence within nucleic acid of a cell, including contacting said cell with:

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

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

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

4) nucleic acid encoding one or more gRNA molecules of the aforementioned gRNA molecule aspects and embodiments, and nucleic acid encoding a Cas9 molecule (e.g., as described herein); or

5) any of 1) to 4), above, and a template nucleic acid (e.g., as described herein);

6) any of 1) to 4) above, and nucleic acid including sequence encoding a template nucleic acid (e.g., as described herein);

7) a composition as described herein (e.g., of any of the aforementioned composition aspects and embodiments); or

8) the vector of any of the aforementioned vector aspects and embodiments.

In embodiments, the gRNA molecule or nucleic acid encoding the gRNA molecule, and the Cas9 molecule or nucleic acid encoding the Cas9 molecule, are formulated in a single composition. In other embodiments, the gRNA molecule or nucleic acid encoding the gRNA molecule, and the Cas9 molecule or nucleic acid encoding the Cas9 molecule, are formulated in more than one composition. In embodiments, the more than one composition are delivered simultaneously or sequentially.

In embodiments, the cell is an animal cell. In embodiments, the cell the cell is a mammalian, primate, or human cell. In embodiments, the cell is a hempatopoietic stem or progenitor cell (HSPC) (e.g., a population of HSPCs). In embodiments, the cell is a CD34+ cell. In embodiments, the cell is a CD34+/CD38−/CD90+/CD45RA− cell. In embodiments, the cell is a CD34+/CD90+/CD49f+ cell. In embodiments, the cell is a CD34+/CD38−/CD90+/CD45RA−/CD49f+ cell. In embodiments, the method includes a population of cells that has been enriched for HSPCs, e.g., for CD34+ cells.

In embodiments, the cell (e.g. population of cells) has been isolated from bone marrow. In embodiments, the cell (e.g. population of cells) has been isolated from mobilized peripheral blood. In embodiments, the cell (e.g. population of cells) has been isolated from umbilical cord blood.

In embodiments, the cell (e.g. population of cells) is autologous with respect to a patient to be administered said cell (e.g. population of cells). In embodiments, the cell (e.g. population of cells) is allogeneic with respect to a patient to be administered said cell (e.g. population of cells).

In embodiments, of the methods described herein, the altering results in an increase of fetal hemoglobin expression in the cell. In embodiments, of the methods described herein, the altering results in a reduction of fetal hemoglobin expression in the cell.

In another aspect, the invention provides a cell, altered by the method of any of the aforementioned method aspects and embodiments. In another aspect, the invention provides a cell, including a first gRNA molecule (e.g., as described herein), or a composition (e.g., as described herein), or a nucleic acid encoding the first gRNA molecule (e.g., as described herein), of any of the previous aspects and embodiments. In embodiments, the cell is an animal cell. In embodiments, the cell the cell is a mammalian, primate, or human cell. In embodiments, the cell is a hematopoietic stem or progenitor cell (HSPC) (e.g., a population of HSPCs). In embodiments, the cell is a CD34+ cell. In embodiments, the cell is a CD34+/CD38−/CD90+/CD45RA− cell. In embodiments, the cell is a CD34+/CD90+/CD49f+ cell. In embodiments, the method includes a population of cells that has been enriched for HSPCs, e.g., for CD34+ cells.

In embodiments, the cell includes, has included, or will include a second gRNA molecule, e.g., as described herein, e.g., of any of the previous gRNA molecule aspects and embodiments, or a nucleic acid encoding the second gRNA molecule, wherein the first gRNA molecule and second gRNA molecule include nonidentical targeting domains.

In embodiments, expression of fetal hemoglobin is increased in the cell, e.g., relative to a cell of the same cell type that has not been modified to include a gRNA molecule (e.g., as described herein). In embodiments, expression of beta globin is decreased in the cell, e.g., relative to a cell of the same cell type that has not been modified to include a gRNA molecule (e.g., as described herein). In embodiments, expression of fetal hemoglobin is increased and expression of beta globin is decreased in the cell, e.g., relative to a cell of the same cell type that has not been modified to include a gRNA molecule (e.g., as described herein).

In an aspect, the cells of the invention (or methods comprising a cell) have been contacted with a stem cell expander, e.g., as described herein, e.g., compound 1, compound 2, compound 3 or compound 4. In an aspect, the cells of the invention (or methods comprising a cell) have been contacted with a stem cell expander, e.g., as described herein, e.g., compound 1, compound 2, compound 3, compound 4 or a combination thereof (e.g., compound 1 and compound 4). In an embodiment, the stem cell expander is compound 4. In an embodiment, the stem cell expander is a combination of compound 1 and compound 4. In embodiments, the contacting is ex vivo.

In another aspect, the invention provides methods of treatment, e.g., methods of treatment of hemoglobinopathies. In an aspect, the invention provides a method of treating a hemoglobinopathy, that includes administering to a patient a cell (e.g., a population of cells) of any of the previous cell or method aspects and embodiments.

In another aspect, the invention provides a method of increasing fetal hemoglobin expression in a mammal, including administering to a patient a cell of any of the previous cell or method aspects and embodiments. In an embodiment, the hemoglobinopathy is beta-thalassemia or sickle cell disease.

In another aspect, the invention provides a guide RNA molecule, e.g., as described herein, for use as a medicament, e.g., for use in the treatment of a disease. In embodiments, the disease is a hemoglobinopathy, e.g., beta-thalassemia or sickle cell disease.

In another aspect, the invention provides a gRNA molecule, e.g., as described herein, e.g., as described in any of the aforementioned gRNA molecule aspects and embodiments, wherein when a CRISPR system including the gRNA is introduced into a cell (e.g., a CD34+ cell, e.g., an HSC), at least about 15% of the indels produced include (a) a frameshift mutation; or (b) a large deletion, relative to unmodified target DNA, as measured by NGS. In embodiments, at least about 25% of the indels produced include (a) a frameshift mutation; or (b) a large deletion, relative to unmodified target DNA, as measured by NGS. In embodiments, 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 indels produced include (a) a frameshift mutation; or (b) a large deletion, relative to unmodified target DNA, as measured by NGS.

In another aspect, the invention provides methods for the ex vivo expansion and modification of HSPCs (e.g., CD34+ cells). In an aspect, the invention provides a method of modifying cells (e.g., a population of cells) including:

(a) providing a population of cells;

(b) expanding said cells ex vivo in the presence of a stem cell expander; and

(c) introducing into said cells a first gRNA molecule (e.g., as described herein, e.g., of any of the aforementioned gRNA molecule aspects and embodiments), a nucleic acid molecule encoding the first gRNA molecule, or a composition (e.g., as described herein, e.g., of any of the aforementioned composition aspects and embodiments);

such that hemoglobin, e.g., fetal hemoglobin, expression is increased in said cells relative to the same cell type which have not been subjected to step (c).

In embodiments, the cells are introduced into a subject in need thereof, e.g., a subject that has a hemoglobinopathy, e.g., sickle cell disease or beta thalassemia.

In embodiments, the cells are or comprise CD34+ cells. In embodiments, the cells are or comprise HSPCs. In embodiments, the cells are isolated from bone marrow, mobilized peripheral blood or umbilical cord blood. In a preferred embodiment, the cells are isolated from bone marrow. In other embodiments, the cells are isolated from mobilized peripheral blood. In aspects, the moblized peripheral blood is isolated from a subject who has been administered a G-CSF. In aspects, the moblized peripheral blood is isolated from a subject who has been administered a moblization agent other than G-CSF, for example, Plerixafor® (AMD3100). In embodiments, the cells are an enriched population of cells.

In embodiments, the stem cell expander is compound 1, compound 2, compound 3, or compound 4, e.g., compound 4. In embodiments, the stem cell expander is compound 1, compound 2, compound 3, compound 4, or a combination thereof (e.g., a combination of compound 1 and compound 4). In embodiments, the cells are contacted with compound 4 at a concentration of from about 1 to about 200 micromolar (uM). In an embodiment, the concentration of compound 4 is about 75 micromolar (uM). In embodiments, the expanding said cells ex vivo in the presence of a stem cell expander occurs for a period of about 1-10 days, e.g., about 1-5 days, e.g., about 2-5 days, e.g., about 4 days.

In embodiments, the cells are autologous to a patient intended to be administered said cells. In embodiments, the cells are allogeneic to a patient intended to be administered said cells.

In embodiments, the expanding of step (b) is further in the presence of thrombopoietin (Tpo), Flt3 ligand (Flt-3L), human stem cell factor (SCF) and human interleukin-6 (IL-6). In embodiments, the thrombopoietin (Tpo), Flt3 ligand (Flt-3L), human stem cell factor (SCF) and human interleukin-6 (IL-6) are each at a concentration of about 50 ng/mL. In embodiments, the thrombopoietin (Tpo), Flt3 ligand (Flt-3L), human stem cell factor (SCF) and human interleukin-6 (IL-6) are each at a concentration of 50 ng/mL.

Additional aspects and embodiments are described below.

In an aspect, the invention provides a gRNA molecule that includes a tracr and crRNA, wherein the crRNA includes a targeting domain that is complementary with a target sequence of a BCL11A gene (e.g., a human BCL11a gene), a BCL11a enhancer (e.g., a human BCL11a enhancer), or a HFPH region (e.g., a human HPFH region).

In embodiments, the target sequence is of the BCL11A gene, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 1 to SEQ ID NO: 85 or SEQ ID NO: 400 to SEQ ID NO: 1231. These embodiments are referred to herein as gRNA molecule embodiment 2.

In other embodiments, the target sequence is of a BCL11a enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 1232 to SEQ ID NO: 1499. These embodiments are referred to herein as gRNA molecule embodiment 3. In preferred embodiments, the target sequence is of a BCL enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 182 to SEQ ID NO: 277 or SEQ ID NO: 334 to SEQ ID NO: 341. These embodiments are referred to herein as gRNA molecule embodiment 4. In more preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 341, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 247, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO: 244, SEQ ID NO: 199, SEQ ID NO: 251, SEQ ID NO: 250, SEQ ID NO: 334, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 336, or SEQ ID NO: 337. These embodiments are referred to herein as gRNA molecule embodiment 5. In still more preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, or SEQ ID NO: 338 (referred to herein as gRNA molecule embodiment 6), for example, includes, e.g., consists of, any one of SEQ ID NO: 248 or SEQ ID NO: 338 (referred to herein as gRNA molecule embodiment 7). In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 248 (referred to herein as gRNA molecule embodiment 8). In embodiments, the targeting domain includes, e.g., consists of, SEQ ID NO: 338 (referred to herein as gRNA molecule embodiment 9).

In other embodiments, the target sequence is of a BCL11a enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 278 to SEQ ID NO: 333 (referred to herein as gRNA molecule embodiment 10). In preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 318, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 294, SEQ ID NO: 310, SEQ ID NO: 319, SEQ ID NO: 298, SEQ ID NO: 322, SEQ ID NO: 311, SEQ ID NO: 315, SEQ ID NO: 290, SEQ ID NO: 317, SEQ ID NO: 309, SEQ ID NO: 289, or SEQ ID NO: 281 (referred to herein as gRNA molecule embodiment 11). In one preferred embodiment, the targeting domain includes, e.g., consists of, SEQ ID NO: 318 (referred to herein as gRNA molecule embodiment 12).

In other embodiments, the target sequence is of a BCL11a enhancer, and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 1596 to SEQ ID NO: 1691 (referred to herein as gRNA molecule embodiment 13). In preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 1683, SEQ ID NO: 1638, SEQ ID NO: 1647, SEQ ID NO: 1609, SEQ ID NO: 1621, SEQ ID NO: 1617, SEQ ID NO: 1654, SEQ ID NO: 1631, SEQ ID NO: 1620, SEQ ID NO: 1637, SEQ ID NO: 1612, SEQ ID NO: 1656, SEQ ID NO: 1619, SEQ ID NO: 1675, SEQ ID NO: 1645, SEQ ID NO: 1598, SEQ ID NO: 1599, SEQ ID NO: 1663, SEQ ID NO: 1677, or SEQ ID NO: 1626 (referred to herein as gRNA molecule embodiment 14).

In other embodiments, the target sequence is of a HFPH region (e.g., a French HPFH region), and the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 86 to SEQ ID NO: 181, SEQ ID NO: 1500 to SEQ ID NO: 1595, or SEQ ID NO: 1692 to SEQ ID NO: 1761 (referred to herein as gRNA molecule embodiment 15). In preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 100, SEQ ID NO: 165, SEQ ID NO: 113, SEQ ID NO: 99, SEQ ID NO: 112, SEQ ID NO: 98, SEQ ID NO: 1580, SEQ ID NO: 106, SEQ ID NO: 1503, SEQ ID NO: 1589, SEQ ID NO: 160, SEQ ID NO: 1537, SEQ ID NO: 159, SEQ ID NO: 101, SEQ ID NO: 162, SEQ ID NO: 104, SEQ ID NO: 138, SEQ ID NO: 1536, SEQ ID NO: 1539, SEQ ID NO: 1585 (referred to herein as gRNA molecule embodiment 16). In still more preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 1505, SEQ ID NO: 1589, SEQ ID NO: 1700, or SEQ ID NO: 1750 (referred to herein as gRNA molecule embodiment 17). In other preferred embodiments, the targeting domain includes, e.g., consists of, any one of SEQ ID NO: 100, SEQ ID NO: 165, or SEQ ID NO: 113 (referred to herein as gRNA molecule embodiment 18).

In embodiments, the gRNA molecule includes a targeting domain that includes, e.g., consists of, 17, 18, 19, 20, 21, 22, 23, or 24, preferably 20, consecutive nucleic acids of any targeting domain sequence described herein, for example a targeting domain sequence of Table 1 or Table 2. In embodiments, the 17, 18, 19, 20, 21, 22, 23, or 24, preferably 20, consecutive nucleic acids of any one of targeting domain sequences described herein, for example a targeting domain sequence of Table 1 or Table 2, are the 17, 18, 19, 20, 21, 22, 23, or 24, preferably 20, consecutive nucleic acids disposed at the 3′ end of the recited targeting domain sequence. In other embodiments, the 17, 18, 19, 20, 21, 22, 23, or 24, preferably 20, consecutive nucleic acids of any one of the targeting domain sequences described herein, for example a targeting domain sequence of Table 1 or Table 2, are the 17, 18, 19, 20, 21, 22, 23, or 24, preferably 20, consecutive nucleic acids disposed at the 5′ end of the recited targeting domain sequence. In other embodiments, the 17, 18, 19, 20, 21, 22, 23, or 24, preferably 20, consecutive nucleic acids of any one of the targeting domain sequences described herein, for example a targeting domain sequence of Table 1 or Table 2, do not include either the 5′ or 3′ nucleic acid of the recited targeting domain sequence.

In embodiments, the gRNA molecule includes a targeting domain that includes, e.g., consists of, 17, 18, 19, or 20, preferably 20, consecutive nucleic acids of any targeting domain sequence described herein, for example a targeting domain sequence of Table 5, Table 6, Table 7, Table 8 or Table 9. In embodiments, the 17, 18, 19, or 20, preferably 20, consecutive nucleic acids of any one of targeting domain sequences described herein, for example a targeting domain sequence of Table 5, Table 6, Table 7, Table 8 or Table 9, are the 17, 18, 19, or 20, preferably 20, consecutive nucleic acids disposed at the 3′ end of the recited targeting domain sequence. In other embodiments, the 17, 18, 19, or 20, preferably 20, consecutive nucleic acids of any one of the targeting domain sequences described herein, for example a targeting domain sequence of Table 5, Table 6, Table 7, Table 8 or Table 9, are the 17, 18, 19, or 20, preferably 20, consecutive nucleic acids disposed at the 5′ end of the recited targeting domain sequence. In other embodiments, the 17, 18, 19, or 20, preferably 20, consecutive nucleic acids of any one of the targeting domain sequences described herein, for example a targeting domain sequence of Table 5, Table 6, Table 7, Table 8 or Table 9, do not include either the 5′ or 3′ nucleic acid of the recited targeting domain sequence.

The following aspects describe features of the gRNA molecule that may be combined with any of the aforementioned aspects and embodiments. In embodiments, the gRNA molecule, including the gRNA molecule of any of the aforementioned aspects and embodiments, include a portion of the crRNA and a portion of the tracr that hybridize to form a flagpole that includes SEQ ID NO: 6584 or 6585. In embodiments, the flagpole further includes a first flagpole extension, located 3′ to the crRNA portion of the flagpole, wherein said first flagpole extension includes SEQ ID NO: 6586. In embodiments, the flagpole further includes (in addition to or in alternative to the first flagpole extension) a second flagpole extension located 3′ to the crRNA portion of the flagpole and, if present, the first flagpole extension, wherein said second flagpole extension includes SEQ ID NO: 6587.

In embodiments, including in any of the aforementioned aspects and embodiments, the tracr includes SEQ ID NO: 6660 or SEQ ID NO: 6661. In embodiments, the tracr includes SEQ ID NO: 7812, optionally further including, at the 3′ end, an additional 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides. In embodiments, the crRNA includes, from 5′ to 3′, [targeting domain]-: a) SEQ ID NO: 6584; b) SEQ ID NO: 6585; c) SEQ ID NO: 6605; d) SEQ ID NO: 6606; e) SEQ ID NO: 6607; f) SEQ ID NO: 6608; or g) SEQ ID NO: 7806.

In embodiments, including in any of the aforementioned aspects and embodiments, the tracr includes, from 5′ to 3′: a) SEQ ID NO: 6589; b) SEQ ID NO: 6590; c) SEQ ID NO: 6609; d) SEQ ID NO: 6610; e) SEQ ID NO: 6660; f) SEQ ID NO: 6661; g) SEQ ID NO: 7812; h) SEQ ID NO: 7807; i) SEQ ID NO: 7808; j) SEQ ID NO: 7809; k) any of a) to j), above, further including, at the 3′ end, at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 uracil (U) nucleotides; 1) any of a) to k), above, further including, at the 3′ end, at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides; or m) any of a) to l), above, further including, at the 5′ end (e.g., at the 5′ terminus), at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides, e.g., 1, 2, 3, 4, 5, 6, or 7 adenine (A) nucleotides.

In embodiments, the targeting domain and the tracr are disposed on separate nucleic acids molecules. In embodiments of such gRNA molecules, the nucleic acid molecule including the targeting domain includes SEQ ID NO: 6607, optionally disposed immediately 3′ to the targeting domain, and the nucleic acid molecule including the tracr includes, e.g., consists of, SEQ ID NO: 6660.

In embodiments, the crRNA portion of the flagpole includes SEQ ID NO: 6607 or SEQ ID NO: 6608.

In embodiments, the tracr includes SEQ ID NO: 6589 or 6590, and optionally, if a first flagpole extension is present, a first tracr extension, disposed 5′ to SEQ ID NO: 6589 or 6590, said first tracr extension including SEQ ID NO: 6591.

In embodiments, including in embodiments of any of the aforementioned aspects and embodiments, the targeting domain and the tracr are disposed on separate nucleic acid molecules. In other embodiments, including in embodiments of any of the aforementioned aspects and embodiments, the targeting domain and the tracr are disposed on a single nucleic acid molecule, for example, the tracr is disposed 3′ to the targeting domain.

In embodiments, when the targeting domain and the tracr are disposed on a single nucleic acid molecule, the gRNA molecule further includes a loop, disposed 3′ to the targeting domain and 5′ to the tracr. In embodiments, the loop includes, e.g., consists of, SEQ ID NO: 6588.

In embodiments, when the targeting domain and the tracr are disposed on a single nucleic acid molecule, the gRNA molecule includes, from 5′ to 3′, [targeting domain]-: (a) SEQ ID NO: 6601; (b) SEQ ID NO: 6602; (c) SEQ ID NO: 6603; (d) SEQ ID NO: 6604; (e) SEQ ID NO: 7811; or (f) any of (a) to (e), above, further including, at the 3′ end, 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides. In embodiments, the gRNA molecule includes, e.g., consists of, said targeting domain and SEQ ID NO: 7811, optionally disposed immediately 3′ to said targeting domain.

In embodiments, including in any of the aforementioned aspects and embodiments, each of the nucleic acid residues of the gRNA molecule is an unmodified A, U, G or C nucleic acid residue and unmodified phosphate bonds between each residue of the nucleic acid molecule(s). In other embodiments, including in any of the aforementioned aspects and embodiments, one, or optionally more than one, of the nucleic acid molecules that make up the gRNA molecule includes: a) one or more, e.g., three, phosphorothioate modifications at the 3′ end of said nucleic acid molecule or molecules; b) one or more, e.g., three, phosphorothioate modifications at the 5′ end of said nucleic acid molecule or molecules; c) one or more, e.g., three, 2′-O-methyl modifications at the 3′ end of said nucleic acid molecule or molecules; d) one or more, e.g., three, 2′-O-methyl modifications at the 5′ end of said nucleic acid molecule or molecules; e) a 2′ O-methyl modification at each of the 4^th-to-terminal, 3^rd-to-terminal, and 2^nd-to-terminal 3′ residues of said nucleic acid molecule or molecules; f) a 2′ O-methyl modification at each of the 4^th-to-terminal, 3^rd-to-terminal, and 2^nd-to-terminal 5′ residues of said nucleic acid molecule or molecules; or f) any combination thereof.

In preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 342; (b) SEQ ID NO: 343; or (c) SEQ ID NO: 1762 (referred to in this summary of invention as gRNA molecule embodiment 41).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 344, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 344, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 345, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 345, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 42).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 347; (b) SEQ ID NO: 348; or (c) SEQ ID NO: 1763 (referred to in this summary of invention as gRNA molecule embodiment 43).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 349, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 349, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 350, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 350, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 44).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 351; (b) SEQ ID NO: 352; or (c) SEQ ID NO: 1764 (referred to in this summary of invention as gRNA molecule embodiment 45).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 353, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 353, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 354, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 354, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 46).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 355; (b) SEQ ID NO: 356; or (c) SEQ ID NO: 1765 (referred to in this summary of invention as gRNA molecule embodiment 47).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 357, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 357, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 358, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 358, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 48).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 359; (b) SEQ ID NO: 360; or (c) SEQ ID NO: 1766 (referred to in this summary of invention as gRNA molecule embodiment 49).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 361, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 361, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 362, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 362, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 50).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 363; (b) SEQ ID NO: 364; or (c) SEQ ID NO: 1767 (referred to in this summary of invention as gRNA molecule embodiment 51).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 365, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 365, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 366, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 366, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 52).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 367; (b) SEQ ID NO: 368; or (c) SEQ ID NO: 1768 (referred to in this summary of invention as gRNA molecule embodiment 53).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 369, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 369, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 370, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 370, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 54).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 371; (b) SEQ ID NO: 372; or (c) SEQ ID NO: 1769 (referred to in this summary of invention as gRNA molecule embodiment 55).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 373, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 373, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 374, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 374, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 56).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 375; (b) SEQ ID NO: 376; or (c) SEQ ID NO: 1770 (referred to in this summary of invention as gRNA molecule embodiment 57).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 377, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 377, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 378, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 378, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 58).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 379; (b) SEQ ID NO: 380; or (c) SEQ ID NO: 1771 (referred to in this summary of invention as gRNA molecule embodiment 59).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 381, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 381, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 382, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 382, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 60).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 383; (b) SEQ ID NO: 384; or (c) SEQ ID NO: 1772 (referred to in this summary of invention as gRNA molecule embodiment 61).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 385, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 385, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 386, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 386, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 62).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 387; (b) SEQ ID NO: 388; or (c) SEQ ID NO: 1773 (referred to in this summary of invention as gRNA molecule embodiment 63).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 389, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 389, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 390, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 390, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 64).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 391; (b) SEQ ID NO: 392; or (c) SEQ ID NO: 1774 (referred to in this summary of invention as gRNA molecule embodiment 65).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 393, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 393, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 394, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 394, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 66).

In other preferred embodiments, the invention provides a gRNA molecule (e.g., that is a sgRNA molecule) including, e.g., consisting of, the sequence: (a) SEQ ID NO: 395; (b) SEQ ID NO: 396; or (c) SEQ ID NO: 1775 (referred to in this summary of invention as gRNA molecule embodiment 67).

In other preferred embodiments, the invention provides gRNA molecule (e.g., that is a dual gRNA molecule) including, e.g., consisting of: (a) a crRNA including, e.g., consisting of, SEQ ID NO: 397, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; (b) a crRNA including, e.g., consisting of, SEQ ID NO: 397, and a tracr including, e.g., consisting of, SEQ ID NO: 346; (c) a crRNA including, e.g., consisting of, SEQ ID NO: 398, and a tracr including, e.g., consisting of, SEQ ID NO: 6660; or (d) a crRNA including, e.g., consisting of, SEQ ID NO: 398, and a tracr including, e.g., consisting of, SEQ ID NO: 346 (referred to in this summary of invention as gRNA molecule embodiment 68).

In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a cell, an indel is formed at or near the target sequence complementary to the targeting domain of the gRNA molecule. In embodiments, including in embodiments comprising a targeting domain complementary to a target sequence of a +58 Enhancer region, the indel does not include a nucleotide of a GATA-1 and/or TAL-1 binding site. In embodiments, the indel does not interfere with the binding of GATA-1 and/or TAL-1 to their binding sites. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a population of cells, an indel is formed at or near the target sequence complementary to the targeting domain of the gRNA molecule in at least about 40%, e.g., at least about 50%, e.g., at least about 60%, e.g., at least about 70%, e.g., at least about 80%, e.g., at least about 90%, e.g., at least about 95%, e.g., at least about 96%, e.g., at least about 97%, e.g., at least about 98%, e.g., at least about 99%, of the cells of the population. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a population of cells, an indel that does not include a nucleotide of a GATA-1 and/or TAL-1 binding site is formed at or near the target sequence complementary to the targeting domain of the gRNA molecule in at least about 20%, e.g., at least about 30%, e.g., at least about 35%, e.g., at least about 40%, e.g., at least about 45%, e.g., at least about 50%, e.g., at least about 55%, e.g., at least about 60%, e.g., at least about 65%, e.g., at least about 70%, e.g., at least about 75%, e.g., at least about 80%, e.g., at least about 85%, e.g., at least about 90%, e.g., at least about 95%, e.g., at least about 99%, of the cells of the population. In embodiments, including in any of the aforementioned aspects and embodiments, the indel is an indel listed in any of FIG. 25, Table 15, Table 26, Table 27 or Table 37. In embodiments, in at least about 30%, e.g., least about 40%, e.g., at least about 50%, e.g., at least about 60%, e.g., at least about 70%, e.g., at least about 80%, e.g., at least about 90%, e.g., at least about 95%, e.g., at least about 96%, e.g., at least about 97%, e.g., at least about 98%, e.g., at least about 99%, of the cells of the population, the indel is an indel listed in any of FIG. 25, Table 15, Table 26, Table 27 or Table 37. In embodiments, the three most frequently detected indels in said population of cells include the indels associated with any gRNA molecule listed in any of FIG. 25, Table 15, Table 26, Table 27 or Table 37. In embodiments, the indel (or pattern of top indels) is as measured by next generation sequencing (NGS), e.g., as described in the art. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a cell, no off-target indels are formed in said cell, e.g., as detectible by next generation sequencing and/or a nucleotide insertional assay, e.g., assayed in an HPCS cell, e.g., a CD34+ cell. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a population of cells, no off-target indel is 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 of the population of cells, e.g., as detectible by next generation sequencing and/or a nucleotide insertional assay, e.g., assayed in a population of HPSC cells, e.g., a population of CD34+ cells.

In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a cell, expression of fetal hemoglobin is increased in said cell or its progeny, e.g., its erythroid progeny, e.g., its red blood cell progeny. In embodiments, expression of fetal hemoglobin is increased in said cell or its progeny, e.g., its erythroid progeny, e.g., its red blood cell progeny, by 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 60%, e.g., at least about 70%, e.g., at least about 80%, e.g., at least about 90%, e.g., at least about 95%, e.g., at least about 96%, e.g., at least about 97%, e.g., at least about 98%, e.g., at least about 99%. In embodiments, said cell or its progeny, e.g., its erythroid progeny, e.g., its red blood cell 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 to about 9 picograms, or from about 9 to about 10 picograms) fetal hemoglobin per cell. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a cell, levels of fetal hemoglobin (e.g., gamma globin) mRNA are increased in said cell or its progeny, e.g., its erythroid progeny, e.g., its red blood cell progeny. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a cell, expression of BCL11a mRNA is decreased in said cell or its progeny, e.g., its erythroid progeny, e.g., its red blood cell progeny. In embodiments, including in any of the aforementioned aspects and embodiments, the invention provides a gRNA molecule, wherein when a CRISPR system (e.g., an RNP as described herein) including the gRNA molecule is introduced into a cell, levels of BCL11a mRNA are decreased in said cell or its progeny, e.g., its erythroid progeny, e.g., its red blood cell progeny.

In any of the aforementioned aspects and embodiments that mention a cell, the cell is (or population of cells includes) a mammalian, primate, or human cell, e.g., is a human cell or population of human cells. In embodiments, the cell is (or population of cells includes) an HSPC, for example, is CD34+, for example, is CD34+CD90+. In embodiments, the cell (or population of cells) is autologous with respect to a patient to be administered said cell. In other embodiments, the cell (or population of cells) is allogeneic with respect to a patient to be administered said cell.

In another aspect, the invention provides a composition including: 1) one or more gRNA molecules (including a first gRNA molecule) described herein, for example, one or more gRNA molecules of any of the previous aspects and embodiments, and a Cas9 molecule, for example, as described herein; 2) one or more gRNA molecules (including a first gRNA molecule) described herein, for example, one or more gRNA molecules of any of the previous aspects and embodiments, and nucleic acid encoding a Cas9 molecule (described herein); 3) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule) described herein, for example, one or more gRNA molecules of any of the previous aspects and embodiments, and a Cas9 molecule (described herein); 4) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule) described herein, for example, one or more gRNA molecules of any of the previous aspects and embodiments, and nucleic acid encoding a Cas9 molecule (described herein); or 5) any of 1) to 4), above, and a template nucleic acid; or 6) any of 1) to 4) above, and nucleic acid including sequence encoding a template nucleic acid.

In preferred embodiments, the invention provides a composition including a first gRNA molecule (e.g., described herein, e.g., a first gRNA molecule of any of the previous gRNA molecule aspects and embodiments), further including a Cas9 molecule (described herein).

In embodiments, the Cas9 molecule is an active or inactive s. pyogenes Cas9. In embodiments, the Cas9 molecule includes SEQ ID NO: 6611. In embodiments, the Cas9 molecule includes, e.g., consists of: (a) SEQ ID NO: 7821; (b) SEQ ID NO: 7822; (c) SEQ ID NO: 7823; (d) SEQ ID NO: 7824; (e) SEQ ID NO: 7825; (f) SEQ ID NO: 7826; (g) SEQ ID NO: 7827; (h) SEQ ID NO: 7828; (i) SEQ ID NO: 7829; (j) SEQ ID NO: 7830; or (k) SEQ ID NO: 7831.

In preferred embodiments, the first gRNA molecule and Cas9 molecule are present in a ribonuclear protein complex (RNP).

In embodiments, the invention provides a composition, e.g., a composition of any of the previous aspects and embodiments, further including 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 a gRNA molecule of a gRNA molecule described herein, e.g., a gRNA molecule of any of the previous gRNA molecule aspects and embodiments, and wherein each gRNA molecule of the composition is complementary to a different target sequence.

In embodiments, 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 within the same gene or region. In embodiments, the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule are complementary to target sequences not more than 20000 nucleotides, not more than 10000 nucleotides, not more than 6000, not more than 5000 nucleotides, not more than 4000, not more than 1000 nucleotides, not more than 500 nucleotides, not more than 400 nucleotides, not more than 300 nucleotides, not more than 200 nucleotides, not more than 100 nucleotides, not more than 90 nucleotides, not more than 80 nucleotides, not more than 70 nucleotides, not more than 60 nucleotides, not more than 50 nucleotides, not more than 40 nucleotides, not more than 30 nucleotides, not more than 20 nucleotides or not more than 10 nucleotides apart.

In other embodiments, 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 sequence within different genes or regions.

In embodiments, including in any of the aforementioned composition aspects and embodiments, the invention provides a composition including a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and second gRNA molecule are:

(a) independently selected from the gRNA molecules of gRNA molecule embodiment 4, and are complementary to different target sequences;

(b) independently selected from the gRNA molecules of gRNA molecule embodiment 5, and are complementary to different target sequences;

c) independently selected from the gRNA molecules of gRNA molecule embodiment 6, and are complementary to different target sequences; or

(d) independently selected from the gRNA molecules of gRNA molecule embodiment 7, and are complementary to different target sequences; or

(e) independently selected from the gRNA molecules of any of gRNA molecule embodiments 41-56, and are complementary to different target sequences.

(a) independently selected from the gRNA molecules of gRNA molecule embodiment 10, and are complementary to different target sequences; or

(b) independently selected from the gRNA molecules of gRNA molecule embodiment 11, and are complementary to different target sequences.

In embodiments, including in any of the aforementioned composition aspects and embodiments, the invention provides a composition, including a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and second gRNA molecule are:

(a) independently selected from the gRNA molecules of gRNA molecule embodiment 13, and are complementary to different target sequences; or

(b) independently selected from the gRNA molecules of gRNA molecule embodiment 14, and are complementary to different target sequences.

(a) independently selected from the gRNA molecules of gRNA molecule embodiment 16, and are complementary to different target sequences;

(b) independently selected from the gRNA molecules of gRNA molecule embodiment 17, and are complementary to different target sequences;

(c) independently selected from the gRNA molecules of gRNA molecule embodiment 18, and are complementary to different target sequences; or

(d) independently selected from the gRNA molecules of any of gRNA molecule embodiments 57-68, and are complementary to different target sequences.