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 eukaryotic cell comprising
 providing to the eukaryotic cell a guide RNA sequence complementary to a target nucleic acid sequence of genomic DNA of the eukaryotic cell, and   providing to the eukaryotic 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 cell by introducing to the cell a nucleic acid encoding the guide RNA,
 wherein the Cas 9 protein is provided to the cell by introducing to the cell a nucleic acid encoding the Cas9 protein, and 
 wherein the 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 eukaryotic cell is a yeast cell, a plant cell or a mammalian cell. 
     
     
         4 . The method of  claim 1  wherein the guide RNA includes between about 10 to about 250 nucleotides. 
     
     
         5 . The method of  claim 1  wherein the guide RNA includes between about 20 to about 100 nucleotides. 
     
     
         6 . The method of  claim 1  wherein the cell is a human cell. 
     
     
         7 . The method of  claim 1  wherein the eukaryotic cell is altered at a plurality of genomic DNA sites by providing to the eukaryotic 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 . A method of altering expression of a gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising:
 a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and   b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,   wherein components (a) and (b) are located on different vectors of the system, whereby the guide RNA targets the target sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered.   
     
     
         9 . The method of  claim 8 , wherein a vector of the system further comprises a nuclear localization signal (NLS). 
     
     
         10 . The method of  claim 8 , wherein the guide RNA comprises a guide sequence fused to a trans-activating cr (tracr) sequence. 
     
     
         11 . The method of  claim 8 , wherein the Cas9 protein is codon optimized for expression in a eukaryotic cell. 
     
     
         12 . The method of  claim 8 , wherein the eukaryotic cell is a mammalian or human cell. 
     
     
         13 . The method of  claim 8 , wherein the expression of the gene product is decreased. 
     
     
         14 . An engineered CRISPR-Cas system comprising:
 a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence of a DNA molecule in a eukaryotic cell that contains the DNA molecule, wherein the DNA molecule encodes and the eukaryotic cell expresses a gene product, and   b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein, wherein components (a) and (b) are located on different vectors of the system,   whereby the guide RNA targets and hybridizes with the target sequence and the Cas9 protein cleaves the DNA molecule,   whereby expression of the gene product is altered.   
     
     
         15 . The system of  claim 14 , wherein the CRISPR-Cas system further comprises a NLS. 
     
     
         16 . The system of  claim 14 , wherein the guide RNA comprises a guide sequence fused to a tracr sequence. 
     
     
         17 . The system of  claim 14 , wherein the Cas9 protein is codon optimized for expression in a eukaryotic cell. 
     
     
         18 . The system of  claim 14 , wherein the eukaryotic cell is a mammalian or human cell. 
     
     
         19 . The system of  claim 14 , wherein the expression of the gene product is decreased. 
     
     
         20 . An engineered Type II CRISPR-Cas system comprising a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell, wherein the DNA molecule encodes and the eukaryotic cell expresses a gene product and the Cas9 protein cleaves the DNA molecule,
 whereby expression of the gene product is altered.   
     
     
         21 . The CRISPR-Cas system of  claim 20 , wherein the CRISPR-Cas system further comprises a NLS. 
     
     
         22 . The CRISPR-Cas system of  claim 20 , wherein the guide RNA comprises a guide sequence fused to a tracr sequence. 
     
     
         23 . The CRISPR-Cas system of  claim 20 , wherein the Cas9 protein is codon optimized for expression in a eukaryotic cell. 
     
     
         24 . The CRISPR-Cas system of  claim 20 , wherein the eukaryotic cell is a mammalian or human cell.