Targeted modification of rat genome

Abstract

Compositions and methods are provided for modifying a rat genomic locus of interest using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Compositions and methods for generating a genetically modified rat comprising one or more targeted genetic modifications in their germline are also provided. Compositions and methods are provided which comprise a genetically modified rat or rat cell comprising a targeted genetic modification in the rat interleukin-2 receptor gamma locus, the rat ApoE locus, the rat Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. The various methods and compositions provided herein allows for these modified loci to be transmitted through the germline.

We claim: 
     
         1 . A method for targeted modification of a genomic locus of interest in a pluripotent rat cell, comprising
 (a) introducing into the pluripotent rat cell a large targeting vector (LTVEC) comprising an insert nucleic acid flanked with a 5′ rat homology arm and a 3′ rat homology arm, wherein the sum total of the 5′ and the 3′ homology arms is at least 10 kb but less than 150 kb; and   (b) identifying a genetically modified pluripotent rat cell comprising the targeted genetic modification at the genomic locus of interest,   wherein the targeted genetic modification is capable of being transmitted through the germline.   
     
     
         2 . The method of  claim 1 , wherein the targeted genetic modification is biallelic. 
     
     
         3 . The method of  claim 1 , wherein the pluripotent rat cell is a rat embryonic stem (ES) cell. 
     
     
         4 . The method of  claim 1 , wherein the pluripotent rat cell is derived from a DA strain or an ACI strain. 
     
     
         5 . The method of  claim 1 , wherein the pluripotent rat cell is characterized by expression of at least one pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof. 
     
     
         6 . The method of  claim 1 , wherein the pluripotent rat cell is characterized by one of more of the following characteristics:
 (a) lack of expression of one or more pluripotency markers comprising c-Myc, Ecat1, and/or Rexo1;   (b) lack of expression of mesodermal markers comprising Brachyury and/or Bmpr2;   (c) lack of expression of one or more endodermal markers comprising Gata6, Sox17 and/or Sox7; or   (d) lack of expression of one or more neural markers comprising Nestin and/or Pax6.   
     
     
         7 . The method of  claim 1 , wherein the sum total of the 5′ and the 3′ homology arms of the LTVEC is from about 10 kb to about 30 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. 
     
     
         8 . The method of  claim 1 , wherein the sum total of the 5′ and the 3′ homology arms of the LTVEC is from about 16 Kb to about 150 Kb. 
     
     
         9 . The method of  claim 1 , wherein the targeted genetic modification comprises:
 (a) a replacement of an endogenous rat nucleic acid sequence with a homologous or an orthologous nucleic acid sequence;   (b) a deletion of an endogenous rat nucleic acid sequence;   (c) a deletion of an endogenous rat nucleic acid sequence,   wherein the deletion ranges from about 5 kb to about 10 kb, from about 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 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) an exogenous nucleic acid sequence ranging from about 5 kb to about 10 kb, from about 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 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;   (e) an exogenous nucleic acid sequence comprising a homologous or an orthologous nucleic acid sequence;   (f) a chimeric nucleic acid sequence comprising a human and a rat nucleic acid sequence;   (g) a conditional allele flanked with site-specific recombinase target sequences; or,   (h) a reporter gene operably linked to a promoter active in a rat cell.   
     
     
         10 . The method of  claim 1 , wherein the genomic locus of interest comprises (i) a first nucleic acid sequence that is complementary to the 5′ rat homology arm; and (ii) a second nucleic acid sequence that is complementary to the 3′ rat homology arm. 
     
     
         11 . The method of  claim 10 , wherein the first and the second nucleic acid sequence is separated by at least 5 kb but less than 3 Mb. 
     
     
         12 . The method of  claim 10 , wherein the first and the second nucleic acid sequence is separated by at least 5 kb but less than 10 kb, at least 10 kb but less than 20 kb, at least 20 kb but less than 40 kb, at least 40 kb but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kb, at least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb, at least about 200 kb but less than about 300 kb, at least about 300 kb but less than about 400 kb, at least about 400 kb but less than about 500 kb, at least about 500 kb but less than about 1 Mb, at least about 1 Mb but less than about 1.5 Mb, at least about 1.5 Mb but less than about 2 Mb, at least about 2 Mb but less than about 2.5 Mb, or at least about 2.5 Mb but less than about 3 Mb. 
     
     
         13 . The method of  claim 1 , wherein introducing step (a) further comprises introducing a second nucleic acid encoding a nuclease agent that promotes a homologous recombination between the targeting construct and the genomic locus of interest in the pluripotent rat cell. 
     
     
         14 . The method of  claim 13 , wherein the nuclease agent comprises
 (a) a chimeric protein comprising a zinc finger-based DNA binding domain fused to a FokI endonuclease; or,   (b) a chimeric protein comprising a Transcription Activator-Like Effector Nuclease (TALEN) fused to a FokI endonuclease.   
     
     
         15 . The method of  claim 1 , wherein introducing step (a) further comprises introducing into the pluripotent rat cell:
 (i) a first expression construct comprising a first promoter operably linked to a first nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein,   (ii) a second expression construct comprising a second promoter operably linked to a genomic target sequence linked to a guide RNA (gRNA),   wherein the genomic target sequence is immediately flanked on the 3′ end by a Protospacer Adjacent Motif (PAM) sequence.   
     
     
         16 . The method of  claim 15 , wherein the genomic locus of interest comprises the nucleotide sequence of SEQ ID NO: 1. 
     
     
         17 . The method of  claim 15 , wherein the gRNA comprises a third nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). 
     
     
         18 . The method of  claim 15 , wherein the Cas protein is Cas9. 
     
     
         19 . The method of  claim 15 , wherein the gRNA comprises:
 (a) the chimeric RNA of the nucleic acid sequence of SEQ ID NO: 2; or,   (b) the chimeric RNA of the nucleic acid sequence of SEQ ID NO: 3.   
     
     
         20 . The method of  claim 17 , wherein the crRNA comprises SEQ ID NO: 4; SEQ ID NO: 5; or SEQ ID NO: 6. 
     
     
         21 . The method of  claim 17 , wherein the tracrRNA comprises SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         22 . A modified rat genomic locus comprising:
 (i) an insertion of a homologous or orthologous human nucleic acid sequence;   (ii) a replacement of an endogenous rat nucleic acid sequence with the homologous or orthologous human nucleic acid sequence; or   (iii) a combination thereof,
 wherein the modified rat genomic locus is capable of being transmitted through the germline. 
   
     
     
         23 . The modified rat genomic locus of  claim 22 , wherein the size of the insertion or replacement is from about 5 kb to about 400 kb. 
     
     
         24 . The rat genomic locus of  claim 22 , wherein the size of the insertion or replacement is from about 5 kb to about 10 kb, from about 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 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. 
     
     
         25 . A method for making a humanized rat, comprising:
 (a) targeting a genomic locus of interest in a pluripotent rat cell with a targeting construct comprising a human nucleic acid to form a genetically modified pluripotent rat cell;   (b) introducing the genetically modified pluripotent rat cell into a host rat embryo; and   (c) gestating the host rat embryo in a surrogate mother; wherein the surrogate mother produces rat progeny comprising a modified genomic locus that comprises:
 (i) an insertion of a human nucleic acid sequence; 
 (ii) a replacement of the rat nucleic acid sequence at the genomic locus of interest with a homologous or orthologous human nucleic acid sequence; 
 (iii) a chimeric nucleic acid sequence comprising a human and a rat nucleic acid sequence; or 
 (iv) a combination thereof, 
   wherein the modified genomic locus is capable of being transmitted through the germline.   
     
     
         26 . The method of  claim 25 , wherein the targeting construct is a large targeting vector (LTVEC), and the sum total of the 5′ and the 3′ homology arms of the LTVEC is at least 10 kb but less than 150 kb. 
     
     
         27 . The method of  claim 26 , wherein the sum total of the 5′ and the 3′ homology arms of the targeting construct is from about 10 kb to about 30 kb, from about 20 kb to 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, or from about 80 kb to about 100 kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb. 
     
     
         28 . The method of  claim 25 , wherein the human nucleic acid sequence is at least 5 kb but less than 400 kb. 
     
     
         29 . The method of  claim 25 , wherein the human nucleic acid sequence is at least 5 kb but less than 10 kb, at least 10 kb but less than 20 kb, at least 20 kb but less than 40 kb, at least 40 kb but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kb, at least 100 kb but less than 150 kb, at least 150 kb but less than 200 kb, at least 200 kb but less than 250 kb, at least 250 kb but less than 300 kb, at least 300 kb but less than 350 kb, or at least 350 kb but less than 400 kb. 
     
     
         30 . The method of  claim 25 , wherein the pluripotent rat cell is a rat embryonic stem (ES) cell. 
     
     
         31 . The method of  claim 25 , wherein the pluripotent rat cell is derived from a DA strain or an ACI strain. 
     
     
         32 . The method of  claim 25 , wherein the pluripotent rat cell is characterized by expression of at least one pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combination thereof. 
     
     
         33 . The method of  claim 25 , wherein the pluripotent rat cell is characterized by one or more of the following features:
 (a) lack of expression of one or more pluripotency markers comprising c-Myc, Ecat1, and/or Rexo1;   (b) lack of expression of one or more mesodermal markers comprising Brachyury and/or Bmpr2;   (c) lack of expression of one or more endodermal markers comprising Gata6, Sox17, and/or Sox7; or   (d) lack of expression of one or more neural markers comprising Nestin and/or Pax6.   
     
     
         34 . A modified rat comprising a humanized genomic locus, wherein the humanized genomic locus comprises:
 (i) an insertion of a homologous or orthologous human nucleic acid sequence;   (ii) a replacement of a rat nucleic acid sequence at an endogenous genomic locus with a homologous or orthologous human nucleic acid sequence;   (iii) a chimeric nucleic acid sequence comprising a human and a rat nucleic acid sequence or,   (iv) a combination thereof,   wherein the humanized genomic locus is capable of being transmitted through the germline.   
     
     
         35 . A rat or rat cell comprising a targeted genetic modification in its genomic locus,
 wherein the genomic locus is an Interleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or a Rag2/Rag1 locus,   wherein the targeted genetic modification comprises:   (a) a deletion of an endogenous rat nucleic acid sequence at the genomic locus;   (b) an insertion of a homologous nucleic acid, an orthologous nucleic acid, or a chimeric nucleic acid comprising a human and a rat nucleic acid sequence, or   (c) a combination thereof,   wherein the targeted genetic modification is transmissible through the germline of the rat or a rat propagated from the rat cell.   
     
     
         36 . A method for modifying a target genomic locus in an Interleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2 locus or a Rag2/Rag1 locus in a pluripotent rat cell, the method comprising:
 (a) introducing into the pluripotent rat cell a targeting vector comprising an insert nucleic acid flanked with 5′ and 3′ rat homology arms homologous to the target genomic locus,   (b) identifying a genetically modified pluripotent rat cell comprising a targeted genetic modification at the target genomic locus,   wherein the targeted genetic modification is capable of being transmitted through the germline of a rat propagated from the pluripotent rat cell.