Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same

Abstract

The invention provides a method of modifying a targeted site of a double stranded DNA, including a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a selected double stranded DNA and a nucleic acid base converting enzyme are linked, with the double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into the targeted site, without cleaving at least one strand of the double stranded DNA in the targeted site.

What is claimed is: 
     
         1 . A method of modifying a targeted site of a double stranded DNA, comprising:
 contacting said double stranded DNA with at least one complex which comprises (i) a nucleic acid base converting enzyme linked to (ii) a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in the targeted site of the double stranded DNA,   thereby to convert one or more nucleotides in the targeted site to one or more different nucleotides or to delete one or more nucleotides in the targeted site or to insert one or more nucleotides into said targeted site, without introducing a double strand break (DSB) in said double stranded DNA in the targeted site,   wherein the nucleic acid sequence-recognizing module is a CRISPR-Cas system, and wherein the CRISPR-Cas system comprises a nickase protein.   
     
     
         2 . The method of  claim 1  which comprises contacting the double stranded DNA with two or more complexes that each comprise a nucleic sequence-recognizing module that specifically binds to a different target nucleotide sequence. 
     
     
         3 . The method of  claim 2 , wherein the different target nucleotide sequences are present in different genes. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid base converting enzyme is a deaminase. 
     
     
         5 . The method of  claim 4 , wherein the deaminase is a cytidine deaminase. 
     
     
         6 . The method of  claim 1 , wherein the step of contacting comprises introducing a nucleic acid encoding the at least one complex into a cell which comprises the double stranded DNA. 
     
     
         7 . The method of  claim 6 , wherein the cell is a prokaryotic cell, an eukaryotic cell, a microbial cell, a plant cell, an insect cell, an animal cell, a vertebrate cell, or a mammalian cell. 
     
     
         8 . A method of modifying a targeted site in double stranded genomic DNA in each of two or more targeted alleles on homologous chromosomes in a polyploid cell, the method comprising:
 contacting said double stranded genomic DNA of the polyploid cell with at least one complex which comprises (i) a nucleic acid base converting enzyme linked to (ii) a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in the targeted site in the double stranded genomic DNA in each of said two or more targeted alleles on homologous chromosomes in the polyploid cell,   thereby to convert one or more nucleotides in said targeted site in the double stranded genomic DNA in each of said two or more targeted alleles on homologous chromosomes to one or more different nucleotides, or to delete one or more nucleotides in said targeted site in the double stranded genomic DNA in each of said two or more targeted alleles on homologous chromosomes, or to insert one or more nucleotides into said targeted site in the double stranded genomic DNA in each of said two or more targeted alleles on homologous chromosomes,   without introducing a double strand break (DSB) in said double stranded genomic DNA, wherein the nucleic acid sequence-recognizing module is a CRISPR-Cas system, and wherein the CRISPR-Cas system comprises a nickase protein.   
     
     
         9 . The method of  claim 6 , wherein the step of introducing the nucleic acid encoding the at least one complex into the cell comprises introducing an expression vector comprising the nucleic acid encoding the at least one complex into the cell, wherein the nucleic acid is under regulation of an inducible regulatory region, the method further comprising a step of inducing expression of the nudeic acid for an expression period to stabilize the conversion of one or more nucleotides in the targeted site to one or more different nucleotides, or the deletion of one or more nucleotides, or the insertion of one or more nucleotides into said targeted site in the double stranded DNA 
     
     
         10 . The method of  claim 9 , wherein the target nucleotide sequence in the targeted site in the double stranded DNA is present in a gene essential for survival of the cell. 
     
     
         11 . A nudeic acid-modifying enzyme complex, comprising:
 a nucleic acid base converting enzyme, linked to   (ii) a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a targeted site of a double stranded DNA,   wherein the nucleic acid sequence-recognizing module is a CRISPR-Cas system comprising either a Cas protein that is incapable of introducing a double strand break (DSB) in double stranded DNA or a Cas protein in which cleavage activity for only one strand of double stranded DNA has been inactivated,   and wherein the complex is capable of converting one or more nucleotides in the targeted site to one or more other nudeotides, or is capable of deleting one or more nucleotides, or is capable of inserting one or more nucleotides into said targeted site, without introducing a double strand break (DSB) in double stranded DNA in the targeted site.   
     
     
         12 . A nucleic add encoding the nucleic acid-modifying enzyme complex of  claim 11 . 
     
     
         13 . The method of  claim 1 , wherein the nickase protein is a Cas9 D10A mutant nickase protein (nCas9(D10A)). 
     
     
         14 . The method of  claim 1 , wherein the nickase protein is a Cas9 H840A mutant nickase protein (nCas9(H840A)). 
     
     
         15 . The nucleic acid-modifying enzyme complex of  claim 11 , wherein only one of two DNA cleavage abilities of the Cas protein is inactivated. 
     
     
         16 . A nucleic acid encoding the nucleic acid-modifying enzyme complex of  claim 15 .