METHOD FOR GENOME EDITING

• US 20120192298A1
• Filed: 07/23/2010
• Published: 07/26/2012
• Est. Priority Date: 07/24/2009
• Status: Abandoned Application

• Current Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.)
• Original Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.)
• Inventors: Weinstein, Edward, Cui, Xiaoxia, Simmons, Phil
• Application Number:
  US13/386,394
• Publication Number:
  US 20120192298A1
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0318 Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention encompasses a method for creating an animal or cell with at least one chromosomal edit. In particular, the invention relates to the use of targeted zinc finger nucleases to edit chromosomal sequences. The invention further encompasses an animal or a cell created by a method of the invention.

Claims

1. A method for editing a chromosomal sequence, the method comprising:

a. introducing into a non-human embryo comprising the chromosomal sequence at least one nucleic acid encoding a zinc finger nuclease, the embryo being other than a zebrafish or fruit fly embryo, the zinc finger nuclease being able to bind a target sequence in the chromosomal sequence and to cleave a cleavage site in the chromosomal sequence, and, optionally,

(i) at least one donor polynucleotide comprising a donor sequence for integration, an upstream sequence, and a downstream sequence, wherein the donor sequence is flanked by the upstream sequence and the downstream sequence, and wherein the upstream sequence and the downstream sequence share substantial sequence identity with either side of the cleavage site, or

(ii) at least one exchange polynucleotide comprising an exchange sequence that is substantially identical to a portion of the chromosomal sequence at the cleavage site and further comprising at least one nucleotide change; and

b. culturing the embryo to allow expression of the zinc finger nuclease such that the zinc finger nuclease introduces a double-stranded break into the chromosomal sequence at the cleavage site, and wherein the double-stranded break is repaired by either

(i) a non-homologous end-joining repair process such that a mutation is introduced into the chromosomal sequence, or

(ii) a homology-directed repair process such that the donor sequence is integrated into the chromosomal sequence or the exchange sequence is exchanged with the portion of the chromosomal sequence.

2. The method of claim 1, wherein the embryo is a mammalian embryo.

3. The method of claim 1, wherein the embryo is a one cell embryo.

4. The method of claim 1, wherein more than one nucleic acid encoding a zinc finger nuclease is introduced into the embryo.

5. The method of claim 1, wherein the nucleic acid encoding a zinc finger nuclease is an RNA.

6. The method of claim 5, wherein the RNA is capped.

7. The method of claim 5, wherein the RNA is polyadenylated.

8. The method of claim 1, wherein more than one of a polynucleotide chosen from the donor polynucleotides, the exchange polynucleotides, or any combination thereof are introduced into the embryo.

11. A non-human animal comprising at least one edited chromosomal sequence, the animal being created by the method of claim 1.

12. The non-human animal of claim 11, wherein the animal is a rodent.

13. The non-human animal of claim 11, wherein the animal is a livestock animal.

14. The non-human animal of claim 11, wherein the animal is a companion animal.

15. A cell comprising at least one edited chromosomal sequence, the cell being derived from an animal created using the method of claim 1.

18. The cell of claim 15, wherein the cell is a cultured cell, a primary cell, or a stem cell.

9-10. -10. (canceled)

16-17. -17. (canceled)

19. A non-human embryo, the embryo being other than a zebrafish or fruit fly embryo and comprising at least one nucleic acid encoding a zinc finger nuclease that is able to bind a target sequence in the chromosomal sequence and cleave a cleavage site in the chromosomal sequence, and, optionally,

(i) at least one donor polynucleotide comprising a donor sequence for integration, an upstream sequence, and a downstream sequence, wherein the donor sequence is flanked by the upstream sequence and the downstream sequence, and wherein the upstream sequence and the downstream sequence share substantial sequence identity with either side of the cleavage site, or

(ii) at least one exchange polynucleotide comprising an exchange sequence that is substantially identical to a portion of the chromosomal sequence at the cleavage site and which further comprises at least one nucleotide change.

20. The embryo of claim 19, wherein the embryo is a one cell embryo.

21. The embryo of claim 19, wherein the embryo is a mammalian embryo.