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.

We claim: 
     
         1 . A method for antibody production, comprising the steps of:
 (a) providing a mouse or progeny thereof developed from the modified mouse ES cell, wherein the modified mouse ES cell has been produced by contacting a mouse ES cell with:
 a Cas9 protein; 
 a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest; 
 a tracrRNA; and 
 an incoming nucleic acid sequence that is flanked by:
 (i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest; and 
 (ii) a 3′ homolog arm that is homologous to a 3′ target sequence at the genomic locus of interest; 
 
 wherein following the contacting step, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising a disruption of a target protein or domain thereof thereby producing a modified mouse ES cell and developing the mouse from the modified mouse ES cell; 
   (b) immunizing the mouse with a predetermined antigen;   (c) removing B lymphocytes from the mouse and selecting one or more B lymphocytes expressing antibodies that bind to the antigen; and   (d) isolating an antibody expressed by the B lymphocytes.   
     
     
         2 . The method of  claim 1 , further comprising a step of producing the modified mouse ES cell wherein the step comprises contacting a mouse ES cell with:
 a Cas9 protein;   a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest;   a tracrRNA; and   an incoming nucleic acid sequence that is flanked by:
 (i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest; and 
 (ii) a 3′ homolog arm that is homologous to a 3′ target sequence at the genomic locus of interest, 
   wherein following the contacting step, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising a disruption of a target protein or domain thereof thereby producing a modified mouse ES cell;   
     
     
         3 . The method of  claim 1 , wherein the incoming nucleic acid is at least 10 kb in size. 
     
     
         4 . The method of  claim 1 , wherein the targeted genomic modification comprises:
 deletion of a region of the genomic locus of interest wherein the deletion is at least 20 kb; and/or   insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 20 kb.   
     
     
         5 . The method of  claim 2 , wherein prior to performing step (a), the mouse ES cell comprises a deletion of one or more mouse antibody gene segments, antibody variable regions, or antibody constant regions 
     
     
         6 . The method of  claim 2 , wherein prior to performing step (a), the mouse ES cell comprises an insertion of one or more human antibody gene segments, antibody variable regions, or antibody constant regions. 
     
     
         7 . The method of  claim 2 , wherein prior to performing step (a), the mouse ES cell comprises a deletion of one or more mouse antibody gene segments, antibody variable regions, or antibody constant regions and an insertion of one or more human antibody gene segments, antibody variable regions, or antibody constant regions. 
     
     
         8 . The method of  claim 1 , further comprising isolating from the B lymphocytes a nucleic acid encoding the antibody. 
     
     
         9 . The method of  claim 1 , further comprising:
 (e) isolating from the B lymphocytes a nucleic acid encoding the antibody.   
     
     
         10 . The method of  claim 9 , further comprising:
 (f) exchanging the heavy chain constant region nucleotide sequence of the nucleic acid encoding the antibody with a nucleotide sequence encoding a human heavy chain constant region; humanised heavy chain constant region; or a non-naturally occurring human constant region.   
     
     
         11 . The method of  claim 9 , further comprising a step of affinity maturing the variable region of the antibody. 
     
     
         12 . The method of  claim 9 , further comprising a step of inserting the isolated nucleic acid into an expression vector or host. 
     
     
         13 . The method of  claim 1 , wherein the antibody expressed by the B lymphocytes is a IgG-type antibody. 
     
     
         14 . The method of  claim 1 , wherein the method further comprises a step of immortalizing the selected B lymphocytes of progeny thereof after step (d). 
     
     
         15 . The method of  claim 1 , wherein the method further comprises a step of producing hybridomas from the selected B lymphocytes of progeny thereof after step (d). 
     
     
         16 . The method of  claim 4 , wherein the targeted genomic modification comprises insertion of one or more of:
 a. one or more human antibody heavy chain variable domains;   b. one or more human antibody kappa light chain variable domains;   c. one or more human antibody lambda light chain variable domains; and   d. one or more human Fc receptor protein or subunit or domain thereof.   
     
     
         17 . The method of  claim 4 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody heavy chain variable domains and insertion of one or more human antibody heavy chain variable domains. 
     
     
         18 . The method of  claim 4 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody kappa light chain variable domains and insertion of one or more human antibody kappa light chain variable domains. 
     
     
         19 . The method of  claim 4 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody lambda light chain variable domains and insertion of one or more human antibody lambda light chain variable domains. 
     
     
         20 . The method of  claim 4 , wherein the targeted genomic modification comprises deletion of one or more mouse Fc receptor protein or subunit or domain thereof and insertion of one or more human Fc receptor protein or subunit or domain thereof. 
     
     
         21 . The method of  claim 4 , wherein the targeted genomic modification comprises insertion of one or more human antibody gene segments, antibody variable regions, or antibody constant regions. 
     
     
         22 . The method of  claim 4 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody gene segments, antibody variable regions, or antibody constant regions and insertion of one or more human antibody gene segments, antibody variable regions, or antibody constant regions. 
     
     
         23 . The method of  claim 1 , wherein the targeted genomic modification comprises a knock-out of a target protein or domain thereof. 
     
     
         24 . The method of  claim 1 , wherein step (a) comprises the use of at least two CRISPR RNAs; each of which hybridizes to a CRISPR target sequence at the same genomic locus of interest. 
     
     
         25 . The method of  claim 24 , wherein an indel is created at the genomic locus, wherein the indel results in a knock-out or disruption of a target protein. 
     
     
         26 . A pharmaceutical composition comprising an isolated antibody produced by the method method of  claim 1 . 
     
     
         27 . A nucleic acid produced by the method of  claim 9 .