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 method of making an indel mutation in a B2M target polynucleotide sequence in a primary somatic cell comprising contacting the cells in the cell line with a nucleic acid sequence encoding a clustered regularly interspersed short palindromic repeats-associated 9 (Cas9) protein and one guide ribonucleic acid sequence that hybridizes to a target site in the B2M target polynucleotide sequence such that an indel mutation in the B2M target polynucleotide sequence occurs, wherein the efficiency of making the indel mutation in the B2M target polynucleotide sequence is at least 4.5%. 
     
     
         2 . The method according to  claim 1 , wherein the Cas9 protein is  Streptococcus pyogenes  Cas9 protein or a functional portion thereof. 
     
     
         3 . The method according to  claim 2 , wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. 
     
     
         4 . The method according to  claim 1 , wherein the Cas9 protein is from any bacterial species or a functional portion thereof. 
     
     
         5 . The method according to  claim 4 , wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. 
     
     
         6 . The method according to  claim 1 , wherein the nucleic acid sequence encoding the Cas9 protein comprises a modified nucleic acid. 
     
     
         7 . The method according to  claim 6 , wherein the guide ribonucleic acid is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thiouridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate. 
     
     
         8 . The method according to  claim 1 , wherein each target site is a 20-nucleotide DNA sequence. 
     
     
         9 . The method according to  claim 1 , wherein each target site is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein. 
     
     
         10 . The method according to  claim 1 , wherein the target site is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by the Cas protein. 
     
     
         11 . The method according to  claim 1 , wherein the target motif is (N) 20 NGG. 
     
     
         12 . The method according to  claim 1 , wherein each target site is G(N) 19 NGG. 
     
     
         13 . The method according to  claim 1 , wherein any of the Cas protein or the ribonucleic acid are expressed from a plasmid.