Compositions and methods for enhancing homologous recombination
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.
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.