Compositions and methods for enhancing homologous recombination

Patent metadata

Field	Value
Publication number	US-10428327-B2
Application number	US-201715605586-A
Country	US
Kind code	B2
Filing date	May 25, 2017
Priority date	May 27, 2016
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

The present disclosure generally relates to compositions and methods for improving the efficiency of homologous recombination. In particular, the disclosure relates to reagents and the use of such reagents.

First claim

What is claimed is:

1. A method for performing homologous recombination, the method comprising:
(a) generating a double-stranded break in a nucleic acid molecule present inside a cell to produce a cleaved nucleic acid molecule, and
(b) contacting the cleaved nucleic acid molecule generated in (a) with a partially double-stranded donor nucleic acid molecule,
wherein the cleaved nucleic acid molecule and the donor nucleic acid molecule each contain matched termini on at least one end,
wherein the matched termini on at least one end of the cleaved nucleic acid molecule and on at least one end the donor nucleic acid molecule is at least ten nucleotides in length,
wherein the matched terminus on at least one end of the cleaved nucleic acid molecule is double-stranded and the matched terminus on at least one end of the donor nucleic acid molecule comprises a 3′ single-stranded overhang.

2. The method of claim 1 , wherein at least one pair of matched termini of the cleaved nucleic acid molecule and the donor nucleic acid molecule share between ten and seventy-five complementary nucleotides.

3. The method of claim 1 , wherein the donor nucleic acid molecule contains one or more terminal nuclease resistant groups in at least one strand of at least one terminus.

4. The method of claim 3 , wherein the donor nucleic acid molecule contains one or more terminal nuclease resistant groups in both strands of both termini.

5. The method of claim 3 , wherein the donor nucleic acid molecule contains a single terminal phosphorothioate linkage in both strands of both termini.

6. The method of claim 3 , wherein the donor nucleic acid molecule contains two terminal phosphorothioate linkage in both strands of both termini.

7. The method of claim 1 , wherein the donor nucleic acid molecule has asymmetric termini.

8. The method of claim 1 , wherein the single-stranded 3′ overhang of the donor nucleic acid molecule is from 10 to 95 nucleotides in length.

9. The method of claim 8 , wherein the 3′ overhang of the donor nucleic acid molecule contains at least one terminal nuclease resistant group.

10. The method of claim 8 , wherein the 3′ overhang of the donor nucleic acid molecule contains two nuclease resistant groups.

11. The method of claim 9 , wherein the nuclease resistant group is a phosphorothioate linkage.

12. The method of claim 1 , wherein the donor nucleic acid molecule comprises two single-stranded 3′ overhangs, each of which is from 10 to 95 nucleotides in length.

Assignees

• Life Technologies Corp

Inventors

• Liang Xiquan
• Potter Robert
• Ravinder Namritha

Classifications

• C12N15/907

  in mammalian cells · CPC title

• C12N2310/152

  on a single-stranded target, e.g. fold-back TFOs · CPC title

• A01K2217/07

  Animals genetically altered by homologous recombination · CPC title

• C12N15/1082 Primary

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title

• C12N2310/533

  having a mismatch or nick in at least one of the strands · CPC title