THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells.

What is claimed is: 
     
         1 . A composition comprising or encoding at least one guide ribonucleic acid sequence consisting of (a) a nucleotide sequence complementary to or identical to a target nucleotide sequence selected from SEQ ID NOs: 1-139 and 304-333, or (b) a nucleotide sequence with a single nucleotide mismatch to the target nucleotide sequence selected from SEQ ID NOs: 1-139 and 304-333. 
     
     
         2 . The composition of  claim 1 , comprising or encoding two guide ribonucleic acid sequences. 
     
     
         3 . The composition of  claim 1 , wherein at least one of the ribonucleic acids of the guide ribonucleic acid is a modified ribonucleic acid. 
     
     
         4 . The composition of  claim 3 , wherein the modified ribonucleic acid is selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate. 
     
     
         5 . The composition of  claim 1 , wherein the target nucleotide sequence is selected from the group consisting of SEQ ID NOs: 304-333. 
     
     
         6 . The composition of  claim 1 , wherein the target nucleotide sequence is selected from the group consisting of SEQ ID NOs: 309, 314, 315, 316, 317, 331, and 332. 
     
     
         7 . The composition of  claim 1 , wherein the target nucleotide sequence is selected from the group consisting of SEQ ID NOs: 1, 4, 13, 14, 18, 21, 22, 26, 27, 32, 33, 37, 58, 64, 66, 72, 73, 79, 82, 83, 84, 85, 86, 87, 89, 93, 94, 112, 113, 114, 117, 120, 305, 308, and 331. 
     
     
         8 . The composition of  claim 1 , further comprising a nucleic acid encoding a Cas protein. 
     
     
         9 . The composition of  claim 8 , further comprising a promoter operably linked to the nucleic acid encoding a Cas protein. 
     
     
         10 . The composition of  claim 9 , wherein the promoter is optimized for increased expression in human stem cells. 
     
     
         11 . The composition of  claim 10 , wherein the promoter is selected from the group consisting of a Cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter. 
     
     
         12 . The composition of  claim 1 , wherein the composition further comprises a nucleic acid encoding a fluorescent protein. 
     
     
         13 . The composition of  claim 1 , wherein the composition comprises a plasmid encoding the at least one guide ribonucleic acid sequence. 
     
     
         14 . A vector comprising a nucleic acid sequence encoding at least one guide ribonucleic acid sequence consisting of (a) a nucleotide sequence complementary to or identical to a target nucleotide sequence selected from SEQ ID NOs: 1-139 and 304-333, or (b) a nucleotide sequence with a single nucleotide mismatch to the target nucleotide sequence selected from SEQ ID NOs: 1-139 and 304-333. 
     
     
         15 . A cell comprising one or two guide ribonucleic acid sequences consisting of (a) a nucleotide sequence complementary to or identical to a target nucleotide sequence selected from SEQ ID NOs: 1-139 and 304-333, or (b) a nucleotide sequence with a single nucleotide mismatch to the target nucleotide sequence selected from SEQ ID NOs: 1-139 and 304-333. 
     
     
         16 . The cell of  claim 15 , further comprising a Cas protein.