Methods, cells & organisms

Abstract

The invention relates to an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (eg, in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location. The technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (eg, produced by NHEJ) in the present invention and thus is more efficient than prior art techniques. The invention also provides sequential insertion and/or deletions using single- or double-stranded DNA cutting.

1 . A method of nucleic acid recombination, the method comprising using Cas endonuclease-mediated nucleic acid cleavage to create first and second breaks in a nucleic acid strand, thereby creating 5′ and 3′ cut ends and a deletion of a nucleotide sequence between the ends, wherein the deletion is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5′ from the 5′ end and a sequence extending 3′ from the 3′ end. 
     
     
         2 . The method of  claim 1 , wherein Cas9 endonuclease is used for Cas endonuclease mediated nucleic acid cleavage. 
     
     
         3 . The method of  claim 1 , wherein the deleted nucleotide sequence is at least 20 nucleotides. 
     
     
         4 . The method of  claim 1 , wherein the deleted nucleotide sequence comprises a regulatory element or encodes all or part of a protein. 
     
     
         5 . The method of  claim 1 , wherein the deleted nucleotide sequence encodes a protein subunit or domain. 
     
     
         6 . The method of  claim 1 , further comprising inserting an insert nucleotide sequence between the cut ends. 
     
     
         7 . The method of  claim 1 , wherein the deletion is performed by carrying out homologous recombination between an incoming nucleic acid comprising an insert nucleotide sequence flanked by the first and second homology arms, wherein the insert nucleotide sequence is inserted between the 5′ and 3′ ends. 
     
     
         8 . The method of  claim 6 , wherein the insert sequence is at least 10 nucleotides long. 
     
     
         9 . The method of  claim 6 , wherein the insert nucleotide sequence comprises a PAM motif. 
     
     
         10 . The method of  claim 6 , wherein the method is carried out in a cell and the insert sequence replaces an orthologous or homologous sequence in the cell. 
     
     
         11 . The method of  claim 1 , wherein the product of the method comprises a nucleic acid strand comprising a PAM motif no more than 10 nucleotides 3′ of the deletion. 
     
     
         12 . The method of  claim 1 , comprising isolating the nucleic acid product of the method or a progeny nucleic strand comprising the deletion. 
     
     
         13 . The method of  claim 1 , wherein the first homology arm comprises a PAM motif. 
     
     
         14 . The method of  claim 1 , wherein the second homology arm comprises a PAM motif. 
     
     
         15 . The method of  claim 13 , wherein the second homology arm comprises a PAM motif. 
     
     
         16 . The method of  claim 1 , wherein Cas endonuclease-mediated cleavage by recognition of GG or NGG PAM motifs is carried out. 
     
     
         17 . The method of  claim 1 , wherein the method is carried out in a cell. 
     
     
         18 . The method of  claim 17 , wherein the cell is a rodent cell. 
     
     
         19 . The method of  claim 1 , wherein the method is carried out in a non-human zygote. 
     
     
         20 . The method of  claim 1 , wherein the non-human zygote is a rodent zygote.