Methods and compositions for targeted genetic modifications and methods of use

Abstract

Methods and compositions are provided for generating targeted genetic modifications on the Y chromosome or a challenging target locus. Compositions include an in vitro culture comprising an XY pluripotent and/or totipotent animal cell (i.e., XY ES cells or XY iPS cells) having a modification that decreases the level and/or activity of an Sry protein; and, culturing these cells in a medium that promotes development of XY F0 fertile females. Such compositions find use in various methods for making a fertile female XY non-human mammal in an F0 generation.

That which is claimed: 
     
         1 . A method for making a mouse XY embryonic stem (ES) cell line capable of producing a fertile XY female mouse in an F0 generation, comprising:
 (a) modifying a mouse XY ES cell to comprise a modification that decreases the level and/or activity of an Sry protein, wherein the mouse XY ES cell comprises a Y chromosome derived from a 129 strain; and   (b) culturing the modified mouse XY ES cell under conditions that allow for making a mouse XY ES cell line capable of producing a fertile XY female mouse in an F0 generation.   
     
     
         2 . The method of  claim 1 , further comprising:
 (c) introducing the modified mouse XY ES cell into a host embryo;   (d) gestating the host embryo; and   (e) obtaining an F0 XY female mouse, wherein upon attaining sexual maturity the F0 XY female mouse is fertile.   
     
     
         3 . The method of  claim 1 , wherein the mouse XY ES cell is a VGF1 mouse ES cell. 
     
     
         4 . The method of  claim 1 , wherein the decreased level and/or activity of the Sry protein results from a targeted genetic modification at the genomic locus comprising the Sry gene. 
     
     
         5 . The method of  claim 4 , wherein the genetic modification in the Sry gene comprises a modification selected from the group consisting of:
 (I) an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides, a knockout, a knockin, a replacement of an endogenous nucleic acid sequence with a homologous, orthologous, or heterologous nucleic acid sequence, or a combination thereof;   (II) an insertion, a deletion, a knockout, a knockin, a point mutation, or a combination thereof;   (III) an insertion of a selectable marker and/or a reporter gene operably linked to a promoter active in the mouse ES cell;   (IV) an insertion of a reporter gene operably linked to the endogenous Sry promoter; and   (V) an insertion of a reporter gene operably linked to the endogenous Sry promoter, wherein the reporter gene encodes the reporter protein LacZ.   
     
     
         6 . The method of  claim 1 , wherein the culturing step comprises culturing the modified mouse XY ES cell in a low-osmolality medium comprising a base medium and supplements suitable for maintaining the modified mouse ES cell in culture, wherein the base medium exhibits an osmolality from about 200 mOsm/kg to less than about 329 mOsm/kg. 
     
     
         7 . The method of  claim 6 , wherein the base medium exhibits one or more of the following characteristics:
 (I) a conductivity of about 11 mS/cm to about 13 mS/cm;   (II) a salt of an alkaline metal and a halide in a concentration of about 50 mM to about 110 mM;   (III) a carbonic acid salt concentration of about 17 mM to about 30 mM; and   (IV) a total alkaline metal halide salt and carbonic acid salt concentration of about 85 mM to about 130 mM.   
     
     
         8 . The method of  claim 1 , wherein upon introduction of the modified mouse XY ES cell into a host embryo and following gestation of the host embryo, at least 80% of the F0 mice are XY females which upon attaining sexual maturity are fertile. 
     
     
         9 . The method of  claim 1 , wherein the mouse XY ES cell comprises a target genomic locus on the Y chromosome comprising a recognition site for a nuclease agent, and wherein the nuclease agent induces a nick or double-strand break at the recognition site. 
     
     
         10 . The method of  claim 9 , further comprising exposing the mouse XY ES cell to the nuclease agent in the presence of a targeting vector comprising an insert polynucleotide, wherein following exposure to the nuclease agent and the targeting vector, the mouse XY ES cell is modified to contain the insert polynucleotide. 
     
     
         11 . The method of  claim 9 , wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), a meganuclease, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA), or an mRNA encoding a nuclease. 
     
     
         12 . The method of  claim 11 , wherein the Cas protein is Cas9, and wherein the guide RNA (gRNA) comprises:
 (a) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) that targets the recognition site, wherein the recognition site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence; and   (b) a trans-activating CRISPR RNA (tracrRNA).   
     
     
         13 . The method of  claim 2 , wherein the F0 XY female mouse is fertile when crossed to a wild-type mouse. 
     
     
         14 . The method of  claim 13 , wherein the wild-type mouse is C57BL/6. 
     
     
         15 . An in vitro culture comprising the mouse XY ES cell line of  claim 1 . 
     
     
         16 . A method of producing a transgenic mouse homozygous for a targeted genetic mutation in the F1 generation, comprising:
 (a) crossing an F0 XY fertile female mouse having a decreased level and/or activity of the Sry protein with an F0 XY male mouse cohort clonal sibling derived from the same ES cell clone, wherein the F0 XY fertile female mouse and the F0 XY male mouse each is heterozygous for the genetic mutation; and   (b) obtaining an F1 progeny mouse that is homozygous for the genetic modification.   
     
     
         17 . A method for modifying a target genomic locus on the Y chromosome in a cell, comprising:
 (a) providing the cell comprising the target genomic locus on the Y chromosome, wherein the target genomic locus comprises a recognition site for a nuclease agent;   (b) introducing into the cell:
 (i) the nuclease agent, wherein the nuclease agent induces a nick or double-strand break at the recognition site; and 
 (ii) a first targeting vector comprising a first insert polynucleotide flanked by first and second homology arms corresponding to first and second target sites located in sufficient proximity to the recognition site; and 
   (c) identifying at least one cell comprising in its genome the first insert polynucleotide integrated at the target genomic locus, wherein the first insert polynucleotide replaces an endogenous nucleic acid sequence at the target genomic locus.   
     
     
         18 . The method of  claim 17 , wherein the sum total of the first homology arm and the second homology arm is at least 4 kb but less than 150 kb, at least 400 bp but less than 1000 bp, or from about 700 bp to about 800 bp. 
     
     
         19 . The method of  claim 17 , wherein the cell is a mammalian cell or a pluripotent cell. 
     
     
         20 . The method of  claim 19 , wherein the mammalian cell is a non-human cell or a rodent cell. 
     
     
         21 . The method of  claim 20 , wherein the rodent is a rat, a mouse, or a hamster. 
     
     
         22 . The method of  claim 19 , wherein the pluripotent cell is an induced pluripotent stem (iPS) cell, a non-human embryonic stem (ES) cell, a rodent ES cell, a mouse ES cell, or a rat ES cell. 
     
     
         23 . The method of  claim 17 , wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), a meganuclease, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA), or an mRNA encoding a nuclease. 
     
     
         24 . The method of  claim 23 , wherein the Cas protein is Cas9, and wherein the guide RNA (gRNA) comprises:
 (a) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) that targets the recognition site, wherein the recognition site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence; and   (b) a trans-activating CRISPR RNA (tracrRNA).   
     
     
         25 . The method of  claim 17 , 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, 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; or
 wherein the deletion is at least 500 kb.