RNA-Guided Human Genome Engineering

Abstract

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

1 . A method of altering a stem cell comprising
 providing to the stem cell a guide RNA sequence complementary to a target nucleic acid sequence of genomic DNA of the stem cell, and   providing to the stem cell a Cas 9 protein that interacts with the guide RNA and cleaves the genomic DNA in a site specific manner.   
     
     
         2 . The method of  claim 1  wherein the guide RNA is provided to the stem cell by introducing to the stem cell a nucleic acid encoding the guide RNA,
 wherein the Cas 9 protein is provided to the stem cell by introducing to the stem cell a nucleic acid encoding the Cas9 protein, and 
 wherein the stem cell produces the guide RNA and the Cas9 protein, the guide RNA binds to complementary genomic DNA and the Cas 9 protein cleaves the genomic DNA in a site specific manner. 
 
     
     
         3 . The method of  claim 1  wherein the stem cell is a human stem cell 
     
     
         4 . The method of  claim 1  wherein the stem cell is an induced pluripotent stem cell. 
     
     
         5 . The method of  claim 1  wherein the guide RNA includes between about 10 to about 250 nucleotides. 
     
     
         6 . The method of  claim 1  wherein the guide RNA includes between about 20 to about 100 nucleotides. 
     
     
         7 . The method of  claim 1  wherein the stem cell is altered at a plurality of genomic DNA sites by providing to the stem cell a plurality of guide RNA sequences complementary to different sites on genomic DNA of the eukaryotic cell,
 providing to the eukaryotic cell a Cas 9 protein that interacts with the plurality of guide RNA sequences and cleaves the genomic DNA in a site specific manner. 
 
     
     
         8 . The method of  claim 1  further including the step of providing to the stem cell a donor sequence and inserting the donor sequence into the genomic DNA. 
     
     
         9 . The method of  claim 1  wherein the Cas 9 protein is human codon optimized. 
     
     
         10 . The method of  claim 1  wherein the Cas 9 protein includes a nuclear localization signal. 
     
     
         11 . The method of  claim 1  wherein the Cas 9 protein is a nickase. 
     
     
         12 . The method of  claim 1  wherein cleaving the genomic DNA in a site specific manner results in nonhomologous end joining. 
     
     
         13 . The method of  claim 1  wherein cleaving the genomic DNA in a site specific manner results in homologous recombination.