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. A protein complex comprising:
(a) a first fusion protein comprising a DNA-binding domain and at least one catalytically inactive FokI cleavage domain or cleavage half-domain, wherein the cleavage domain or cleavage half-domain comprises a mutation at one or more of amino acid residues 450, 467 and 469; and
(b) a second fusion protein comprising a zinc finger domain and a FokI catalytically active cleavage half domain,
wherein the catalytically inactive FokI cleavage half domain forms a heterodimer with the catalytically active FokI cleavage half-domain of the second fusion protein and further wherein the protein complex induces a single-stranded break in a target double-stranded sequence.
2. A composition comprising one or more polynucleotides encoding the fusion proteins of the protein complex of claim 1 .
3. An isolated cell or cell line comprising a protein complex according to claim 1 .
4. A method of generating a single-stranded break in a target double-stranded sequence in a cell, the method comprising:
providing a composition according to claim 2 , wherein the heterodimer generates a single-stranded break in the target double-stranded sequence.
5. The method of claim 4 , wherein the target sequence is in cellular chromatin.
6. A method of inserting an exogenous sequence into a region of interest in a cell, the method comprising
generating a single-stranded break in the region of interest according to the method of claim 4 ; and
providing an exogenous sequence that is inserted into the region of interest following generation of a single-stranded break.
7. The method of claim 6 , wherein the exogenous sequence replaces a wild-type genomic sequence.
8. A method of inactivating a target sequence in a region of interest of a cell, the method comprising
generating a single-stranded break in the region of interest according to the method of claim 4 , and
providing an exogenous sequence that replaces the wild-type sequence in the region of interest and wherein the exogenous sequence partially or fully inactivates the target sequence.