Large Gene Excision and Insertion

Abstract

Methods of simultaneously excising large nucleic acid sequences from a target nucleic acid and inserting large foreign nucleic sequences into the target nucleic acid sequence using DNA binding protein nucleases are described.

1 . A method of altering a target nucleic acid in a cell comprising introducing into the cell one or more first foreign nucleic acids encoding two or more guide RNA sequences complementary to DNA, wherein the DNA includes the target nucleic acid,
 introducing into the cell a second foreign nucleic acid encoding an RNA guided DNA binding protein of a Type II CRISPR System that binds to the DNA and is guided by the two or more guide RNA sequences,   introducing into the cell an exogenous nucleic acid sequence to be included into the target nucleic acid sequence,   wherein the two or more guide RNA sequences and the RNA guided DNA binding protein of a Type II CRISPR System are expressed,   wherein the two or more guide RNA sequences and the RNA guided DNA binding protein of a Type II CRISPR System co-localize to the DNA and wherein the RNA guided DNA binding protein of a Type II CRISPR System creates two or more double stranded breaks to remove a first nucleic acid sequence of interest and wherein the exogenous nucleic acid sequence is inserted between the two break points of the target nucleic acid,   wherein the first nucleic acid sequence of interest and the exogenous nucleic acid sequence are greater than 1000 base pairs in length, and   wherein the exogenous nucleic acid sequence to be included into the target nucleic acid sequence is flanked by sequences complementary to the area around the first nucleic acid sequence of interest.   
     
     
         2 . The method of  claim 1  wherein the RNA guided DNA binding protein of a Type II CRISPR System is a Cas protein. 
     
     
         3 . The method of  claim 1  wherein the exogenous nucleic acid sequence is between greater than 1000 base pairs and about 100,000 base pairs in length. 
     
     
         4 . The method of  claim 1  wherein the first nucleic acid sequence of interest is between greater than 1000 base pairs and about 10,000 base pairs in length. 
     
     
         5 . The method of  claim 1  wherein the cell is a eukaryotic cell. 
     
     
         6 . The method of  claim 1  wherein the cell is a yeast cell, a plant cell or an animal cell. 
     
     
         7 . The method of  claim 1  wherein the two or more guide RNAs each comprise between about 10 to about 500 nucleotides. 
     
     
         8 . The method of  claim 1  wherein the two or more guide RNAs each comprise is between about 20 to about 100 nucleotides. 
     
     
         9 . The method of  claim 1  wherein the two ene or more guide RNAs each comprise a crRNA. 
     
     
         10 . The method of  claim 1  wherein the two or more guide RNAs each comprise a tracrRNA-crRNA fusion. 
     
     
         11 . The method of  claim 1  wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. 
     
     
         12 . The method of  claim 1  wherein the exogenous nucleic acid sequence is inserted into the target nucleic acid sequence by homologous recombination. 
     
     
         13 . The method of  claim 1  wherein the exogenous nucleic acid sequence is inserted into the target nucleic acid sequence by nonhomologous end joining. 
     
     
         14 . The method of  claim 1  wherein the RNA guided DNA binding protein of a Type II CRISPR System is a Cas9 protein. 
     
     
         15 . The method of  claim 1  wherein the cell is a human cell. 
     
     
         16 . The method of  claim 1  wherein the cell is a human induced pluripotent stem cell. 
     
     
         17 . A CRISPR system for altering a target nucleic acid in a cell comprising
 one or more first foreign nucleic acids encoding two guide RNA sequences that are complementary to DNA, wherein the two guide RNA sequences define a target nucleic acid which is greater than 1000 base pairs in length,   a second foreign nucleic acid encoding a Cas9 protein, and   an exogenous nucleic acid sequence greater than 1000 base pairs in length and is flanked by sequences complementary to the area around the target nucleic acid.   
     
     
         18 . The CRISPR system of  claim 17  wherein the exogenous nucleic acid is between greater than 1000 base pairs and about 100,000 base pairs in length. 
     
     
         19 . The CRISPR system of  claim 17  wherein the first nucleic acid sequence of interest is between greater than 1000 base pairs and about 10,000 base pairs in length. 
     
     
         20 . The CRISPR system of  claim 17  wherein the cell is a eukaryotic cell. 
     
     
         21 . The CRISPR system of  claim 17  wherein the cell is a yeast cell, a plant cell or an animal cell. 
     
     
         22 . The CRISPR system of  claim 17  wherein the cell is a human cell. 
     
     
         23 . The CRISPR system of  claim 17  wherein the cell is a human induced pluripotent stem cell. 
     
     
         24 . The CRISPR system of  claim 17  wherein the two guide RNA sequences each is a tracrRNA-crRNA fusion. 
     
     
         25 . The CRISPR system of  claim 17  wherein the two guide RNA sequences each is a single guide RNA (sgRNA). 
     
     
         26 . The CRISPR system of  claim 17  wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. 
     
     
         27 . A cell comprising the CRISPR system of  claim 17 . 
     
     
         28 . The cell of  claim 27  wherein the cell is a eukaryotic cell. 
     
     
         29 . The cell of  claim 27  wherein the cell is a yeast cell, a plant cell or an animal cell. 
     
     
         30 . The cell of  claim 27  wherein the cell is a human cell. 
     
     
         31 . The cell of  claim 27  wherein the cell is a human induced pluripotent stem cell.