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.

1 .- 56 . (canceled) 
     
     
         57 . A method for modifying a target genomic locus on the Y chromosome in a cell, comprising:
 (a) providing a population of cells comprising a target genomic locus on the Y chromosome;   (b) introducing into the population of cells of step (a) a large targeting vector comprising an insert polynucleotide flanked by first and second homology arms corresponding to first and second target sites located in the target genomic locus on the Y chromosome, wherein the sum total of the first and second homology arms is from 10 kb to 200 kb, and wherein the large targeting vector undergoes homologous recombination with the target genomic locus on the Y chromosome; and   (c) identifying a cell from step (b) comprising in its genome the insert polynucleotide integrated at the target genomic locus on the Y chromosome.   
     
     
         58 . The method of  claim 57 , wherein the cells in step (a) are mammalian cells. 
     
     
         59 . The method of  claim 58 , wherein the cells in step (a) are mouse cells. 
     
     
         60 . The method of  claim 59 , wherein the cells in step (a) are mouse embryonic stem (ES) cells. 
     
     
         61 . The method of  claim 57 , wherein the target genomic locus on the Y chromosome is repeat-rich. 
     
     
         62 . The method of  claim 57 , wherein the target genomic locus on the Y chromosome is a Sry gene, a Uty gene, an Eif2s3y gene, a Ddx3y gene, a Ube1y gene, a Tspy gene, a Usp9y gene, a Zfy1 gene, a Zfy2 gene, or a region encompassing a Kdm5d gene, the Eif2s3y gene, the Tspy gene, the Uty gene, the Ddx3y gene, and the Usp9y gene. 
     
     
         63 . The method of  claim 57 , wherein the target genomic locus on the Y chromosome comprises a Sry gene. 
     
     
         64 . The method of  claim 57 , wherein the cells in step (a) are in a culture comprising a DMEM base medium, wherein the DMEM base medium is not a KO-DMEM medium. 
     
     
         65 . The method of  claim 57 , wherein the cells in step (a) are in a culture comprising a DMEM base medium, wherein the DMEM base medium is not a KO-DMEM medium, and
 wherein the cells in step (a) are mouse embryonic stem (ES) cells.   
     
     
         66 . The method of  claim 57 , wherein the first homology arm is from 5 kb to 100 kb and/or the second homology arm is from 5 kb to 100 kb. 
     
     
         67 . The method of  claim 57 , wherein the large targeting vector is from 15 kb to 300 kb. 
     
     
         68 . The method of  claim 57 , wherein the insert polynucleotide is from 5 kb to 400 kb. 
     
     
         69 . The method of  claim 57 , wherein the insert polynucleotide comprises a conditional allele, a polynucleotide encoding a selection marker, a reporter gene, or a nucleic acid flanked by site-specific recombination target sequences. 
     
     
         70 . The method of  claim 57 , wherein step (b) further comprises introducing into the population of cells a nuclease agent or a polynucleotide encoding the nuclease agent, wherein the nuclease agent induces a nick or double-strand break at a recognition site in the target genomic locus on the Y chromosome. 
     
     
         71 . The method of  claim 70 , wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated 9 (Cas9) protein and a guide RNA (gRNA) comprising: (1) a CRISPR RNA (crRNA) that targets the recognition site, wherein the recognition site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence; and (2) a trans-activating CRISPR RNA (tracrRNA). 
     
     
         72 . The method of  claim 71 , wherein the nuclease agent is the Cas9 protein and the gRNA. 
     
     
         73 . The method of  claim 70 , wherein step (b) comprises introducing the polynucleotide encoding the nuclease agent. 
     
     
         74 . The method of  claim 70 , wherein step (b) comprises introducing the nuclease agent. 
     
     
         75 . The method of  claim 57 , wherein the cell in step (c) is identified using a modification-of-allele assay. 
     
     
         76 . The method of  claim 57 , wherein the cells in step (a) are in a culture comprising a DMEM base medium, wherein the DMEM base medium is not a KO-DMEM medium,
 wherein the cells in step (a) are mouse embryonic stem (ES) cells,   wherein step (b) further comprises introducing into the population of cells a nuclease agent or a polynucleotide encoding the nuclease agent, wherein the nuclease agent induces a nick or double-strand break at a recognition site in the target genomic locus on the Y chromosome, and   wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated 9 (Cas9) protein and a guide RNA (gRNA) comprising: (1) a CRISPR RNA (crRNA) that targets the recognition site, wherein the recognition site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence; and (2) a trans-activating CRISPR RNA (tracrRNA).