Methods and compositions for targeted genetic modifications and methods of use

Patent metadata

Field	Value
Publication number	US-10793874-B2
Application number	US-201815902514-A
Country	US
Kind code	B2
Filing date	Feb 22, 2018
Priority date	Jun 26, 2014
Publication date	Oct 6, 2020
Grant date	Oct 6, 2020

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.

First claim

That which is claimed:

1. A method for modifying a target genomic locus on the Y chromosome in a mouse embryonic stem (ES) cell, comprising:
(a) providing a mouse ES cell comprising a target genomic locus on the Y chromosome, wherein the target genomic locus on the Y chromosome comprises a recognition site for a nuclease agent, and wherein the mouse ES cell is in a culture comprising a DMEM base medium, wherein the DMEM base medium is not a KO-DMEM base medium;
(b) introducing into the mouse ES cell of step (a):
(i) the nuclease agent or a polynucleotide encoding the nuclease agent, wherein the nuclease agent induces a nick or double-strand break at the recognition site, 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); and
(ii) 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 homology arm and the second homology arm is at least 10 kb, and wherein the large targeting vector undergoes homologous recombination with the target genomic locus on the Y chromosome; and

(c) identifying at least one mouse ES cell from step (b) comprising in its genome the insert polynucleotide integrated at the target genomic locus on the Y chromosome, wherein integration of the insert polynucleotide introduces a genetic modification comprising replacement of an endogenous nucleic acid sequence with an exogenous nucleic acid sequence at the target genomic locus on the Y chromosome.

2. The method of claim 1 , wherein the sum total of the first homology arm and the second homology arm is less than 150 kb.

3. The method of claim 1 , wherein step (b) comprises introducing the polynucleotide encoding the nuclease agent, wherein the polynucleotide is an mRNA encoding the nuclease agent.

4. The method of claim 1 , wherein the nuclease agent is the Cas9 protein and the gRNA.

5. The method of claim 1 , wherein the replaced endogenous nucleic acid sequence ranges from about 5 kb to about 3 Mb.

6. The method of claim 1 , wherein the replaced endogenous nucleic acid sequence is at least 500 kb.

7. The method of claim 1 , wherein the insert polynucleotide is from about 5 kb to about 400 kb in length.

8. The method of claim 1 , wherein the insert polynucleotide comprises a conditional allele, a polynucleotide encoding a selection marker, or a reporter gene.

9. The method of claim 1 , wherein the insert polynucleotide comprises a nucleic acid flanked by site-specific recombination target sequences.

10. The method of claim 1 , wherein the modification comprises a domain swap, an exon swap, an intron swap, a regulatory sequence swap, a gene swap, or a combination thereof.

11. The method of claim 1 , wherein the modification comprises a replacement of the endogenous nucleic acid sequence with a homologous or an orthologous nucleic acid sequence.

12. The method of claim 1 , wherein the target genomic locus on the Y chromosome is the Sry gene, the Uty gene, the Eif2s3y gene, the Ddx3y gene, the Ube1y gene, the Tspy gene, the Usp9y gene, the Zfy1 gene, the Zfy2 gene, or the region encompassing the Kdm5d, Eif2s3y, Tspy, Uty, Ddx3y, and Usp9y genes.

13. The method of claim 1 , wherein the target genomic locus on the Y chromosome comprises the Sry gene.

Assignees

• Regeneron Pharma

Inventors

• Frendewey David
• Droguett Gustavo
• Gagliardi Anthony
• Kuno Junko
• Auerbach Wojtek
• Valenzuela David

Classifications

• C12N2517/00

  Cells related to new breeds of animals · CPC title

• C12N2500/60

  Buffer, e.g. pH regulation, osmotic pressure · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N15/8509 Primary

  for producing genetically modified animals, e.g. transgenic · CPC title