Methods and compositions for target DNA modification
View Patent ↗The disclosure provides compositions and methods for increasing efficiency of Cas9-mediated target DNA modification. Specifically, the disclosure provides compositions and methods for carrying out site-directed modification of a target DNA, the methods comprising contacting the target DNA with: a) a complex comprising a Cas9 polypeptide and a guide RNA, and b) a Rad51 polypeptide. The site-directed modification of a target DNA can be carried out in a living cell in vitro, in a living cell in vivo, or in a cell-free system in vitro.
1. A method for increasing the efficiency of site specific cleavage of a target DNA, the method comprising contacting the target DNA with:
a) a complex comprising a Cas9 polypeptide and a guide RNA, wherein the guide RNA comprises a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA and a second segment that binds to the Cas9 polypeptide; and
b) an amount of a Rad51 polypeptide sufficient to enhance binding of the first segment to the target DNA.
2. The method of claim 1 , wherein the method is an in vitro method.
3. The method of claim 1 , wherein the method is an in vivo method and wherein the target DNA is comprised within a living cell.
4. The method of claim 3 , wherein the Rad51 polypeptide is heterologous to the cell.
5. The method of claim 3 , wherein the cell modified in the in vivo method has been genetically modified to comprise a heterologous nucleic acid that comprises a nucleotide coding sequence encoding the Rad51 polypeptide.
6. The method of claim 5 , wherein the nucleotide sequence is operably linked to a promoter that is functional in the cell.
7. The method of claim 5 , wherein said genetic modification provides for a level of Rad51 that is at least 10% higher than the level of Rad51 polypeptide in a control cell not genetically modified with a heterologous nucleic acid that comprises a nucleotide sequence encoding the Rad51 polypeptide.