Methods and compositions for the targeted modification of a genome

Abstract

Compositions and methods are provided for modifying a genomic locus of interest in a eukaryotic cell, a mammalian cell, a human cell or a non-human mammalian cell using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Further methods combine the use of the LTVEC with a CRISPR/Cas system. Compositions and methods for generating a genetically modified non-human animal comprising one or more targeted genetic modifications in their germline are also provided.

We claim: 
     
         1 . An in vitro method for modifying a genome at a genomic locus of interest in a rodent cell, comprising:
 contacting the rodent cell with a Cas9 protein, a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest, a tracrRNA, and a large targeting vector (LTVEC) that is at least 10 kb in size and comprises an insert nucleic acid 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′ homology arm that is homologous to a 3′ target sequence at the genomic locus of interest,   wherein following contacting the rodent cell with the Cas9 protein, the CRISPR RNA, and the tracrRNA in the presence of the LTVEC, the genome of the rodent cell is modified to comprise a targeted genetic modification comprising deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb and/or insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 30 kb.   
     
     
         2 . The method of  claim 1 , wherein the CRISPR RNA and the tracrRNA are introduced as a single nucleic acid molecule comprising the CRISPR RNA and the tracrRNA. 
     
     
         3 . The method of  claim 2 , wherein the single nucleic acid molecule comprises the CRISPR RNA and the tracrRNA fused together in the form of a single guide RNA (sgRNA). 
     
     
         4 . The method of  claim 1 , wherein the CRISPR RNA and the tracrRNA are introduced separately. 
     
     
         5 . The method of  claim 1 , wherein:
 (a) the Cas9 protein is introduced in the form of a protein, a messenger RNA (mRNA) encoding the Cas9 protein, or a DNA encoding the Cas9 protein;   (b) the CRISPR RNA is introduced in the form of an RNA or a DNA encoding the CRISPR RNA; and   (c) the tracrRNA is introduced in the form of an RNA or a DNA encoding the tracrRNA.   
     
     
         6 . The method of  claim 5 , wherein the Cas9 protein, the CRISPR RNA, and the tracrRNA are introduced as a protein-RNA complex. 
     
     
         7 . The method of  claim 5 , wherein:
 (a) the DNA encoding the Cas9 protein is in the form of a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding the Cas9 protein;   (b) the DNA encoding the CRISPR RNA is in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding the CRISPR RNA; and   (c) the DNA encoding the tracrRNA is in the form of a third expression construct comprising a third promoter operably linked to a third nucleic acid encoding the tracrRNA;   wherein the first, second, and third promoters are active in the rodent cell, and   wherein the first, second, and third expression constructs are on a single nucleic acid molecule or on multiple nucleic acid molecules.   
     
     
         8 . The method of  claim 5 , wherein:
 (a) the DNA encoding the Cas9 protein is in the form of a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding the Cas9 protein; and   (b) the DNA encoding the CRISPR RNA and the DNA encoding the tracrRNA are in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a gRNA comprising the CRISPR RNA and the tracrRNA;   wherein the first and second promoters are active in the rodent cell, and   wherein the first and second expression constructs are on a single nucleic acid molecule or on separate nucleic acid molecules.   
     
     
         9 . The method of  claim 1 , wherein the CRISPR target sequence is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence. 
     
     
         10 . The method of  claim 1 , wherein the targeted genetic modification comprises simultaneous deletion of an endogenous nucleic acid sequence at the genomic locus of interest and insertion of the insert nucleic acid at the genomic locus of interest. 
     
     
         11 . The method of  claim 10 , wherein the deleted endogenous nucleic acid sequence is from 30 kb to about 110 kb, and the insert nucleic acid is from about 40 kb to about 140 kb. 
     
     
         12 . The method of  claim 1 , wherein the targeted genetic modification is a biallelic genetic modification. 
     
     
         13 . The method of  claim 12 , wherein the biallelic genetic modification comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid at the genomic locus of interest in two homologous chromosomes. 
     
     
         14 . The method of  claim 12 , wherein the modified rodent cell is compound heterozygous or hemizygous at the genomic locus of interest. 
     
     
         15 . The method of  claim 14 , wherein the targeted genetic modification at the genomic locus of interest in one chromosome comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid. 
     
     
         16 . The method of  claim 15 , wherein the targeted genetic modification comprises: (1) deletion of an endogenous nucleic acid sequence at the genomic locus of interest in first and second homologous chromosomes; and (2) insertion of the insert nucleic acid into the genomic locus of interest in the first homologous chromosome and disruption of the genomic locus of interest in the second homologous chromosome. 
     
     
         17 . The method of  claim 1 , wherein the targeted genetic modification comprises:
 (a) replacement of an endogenous nucleic acid sequence with a homologous or an orthologous nucleic acid sequence;   (b) deletion of an endogenous nucleic acid sequence;   (c) deletion of an endogenous nucleic acid sequence,   wherein the deletion ranges from at least 30 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200 kb, from about 200 kb to about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb;   (d) insertion of an exogenous nucleic acid sequence;   (e) insertion of an exogenous nucleic acid sequence ranging from at least 30 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb;   (f) insertion of an exogenous nucleic acid sequence comprising a homologous or an orthologous nucleic acid sequence;   (g) insertion of a chimeric nucleic acid sequence comprising a human and a non-human nucleic acid sequence;   (h) insertion of a conditional allele flanked by site-specific recombinase target sequences;   (i) insertion of a selectable marker or a reporter gene operably linked to a promoter active in the rodent cell; or   (j) a combination thereof.   
     
     
         18 . The method of  claim 1 , wherein the sum total of the 5′ and the 3′ homology arms of the LTVEC is from 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb. 
     
     
         19 . The method of  claim 1 , wherein the LTVEC is at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, or at least 200 kb; or
 wherein the targeted genetic modification comprises deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb, and the LTVEC is at least 15 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, or at least 200 kb.   
     
     
         20 . The method of  claim 1 , wherein the targeted genetic modification comprises insertion of the insert nucleic acid,
 wherein the insert nucleic acid is at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, or at least 300 kb; or   wherein the targeted genetic modification further comprises deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb, and the insert nucleic acid is at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, or at least 300 kb.   
     
     
         21 . The method of  claim 1 , wherein the insert nucleic acid is from about 40 kb to about 140 kb. 
     
     
         22 . The method of  claim 1 , wherein the targeted genetic modification comprises deletion of a region of the genomic locus of interest,
 wherein the deletion is at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, or at least 300 kb; or   wherein the targeted genetic modification further comprises insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 30 kb, and the deletion is at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at least 150 kb, at least 200 kb, at least 250 kb, or at least 300 kb.   
     
     
         23 . The method of  claim 1 , wherein the region of the genomic locus of interest deleted is from about 30 kb to about 110 kb. 
     
     
         24 . The method of  claim 1 , wherein the targeted genetic modification comprises deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb and insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 30 kb. 
     
     
         25 . The method of  claim 1 , wherein the rodent cell is a rat cell. 
     
     
         26 . The method of  claim 1 , wherein the rodent cell is a mouse cell. 
     
     
         27 . The method of  claim 1 , wherein the rodent cell is a rodent pluripotent cell. 
     
     
         28 . The method of  claim 27 , wherein the rodent pluripotent cell is a rodent embryonic stem (ES) cell. 
     
     
         29 . The method of  claim 28 , wherein the rodent pluripotent cell is a mouse or rat embryonic stem (ES) cell. 
     
     
         30 . The method of  claim 29 , wherein the rodent pluripotent cell is a rat ES cell.