Methods and compositions for targeted single-stranded cleavage and targeted integration
Disclosed herein are methods and compositions for generating a single-stranded break in a target sequence, which facilitates targeted integration of one or more exogenous sequences.
1. An artificial nuclease comprising
(i) first and second cleavage domains from an endonuclease, wherein the first cleavage domain is catalytically inactive and the second cleavage domain is catalytically active; and
(ii) a DNA-binding molecule that is heterologous to the first and second cleavage domains, and further wherein the DNA-binding molecule of the nuclease binds to a target sequence in a double-stranded genome and the nuclease induces a site-specific single-stranded break at or near the target sequence in the double-stranded genome such that the double-stranded genome is modified.
2. The nuclease of claim 1 , wherein the modification is selected from the group consisting of insertions, deletions and combinations thereof.
3. The nuclease of claim 2 , wherein modification results in inactivation of an endogenous gene.
4. The nuclease of claim 2 , wherein the modification comprises targeted integration of an exogenous sequence.
5. A composition comprising the nuclease of claim 1 .
6. An isolated cell or cell line comprising a nuclease of claim 1 .
7. A method of generating a single-stranded break in a target double-stranded sequence in cellular chromatin of a cell, the method comprising:
providing a nuclease according to claim 1 to the cell such that the nuclease generates a single-stranded break in the target double-stranded sequence in cellular chromatin.
8. The method of claim 7 , further comprising providing an exogenous sequence to the cell such that the exogenous sequence is inserted into the cellular chromatin at or near the single-stranded break.
9. The method of claim 8 , wherein the exogenous sequence replaces a wild-type genomic sequence.
10. The method of claim 7 , wherein a target sequence in the cellular chromatin is inactivated.
11. The method of claim 7 , further comprising providing a second nuclease that makes a second single-stranded break in the double-stranded cellular chromatin.
12. A cell made by the method of claim 7 .