Methods and compositions for targeted cleavage and recombination
Disclosed herein are methods and compositions for targeted cleavage of a genomic sequence, targeted alteration of a genomic sequence, and targeted recombination between a genomic region and an exogenous polynucleotide homologous to the genomic region. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain and polynucleotides encoding same. Methods for targeted cleavage include introduction of such fusion proteins, or polynucleotides encoding same, into a cell. Methods for targeted recombination additionally include introduction of an exogenous polynucleotide homologous to a genomic region into cells comprising the disclosed fusion proteins.
1. A method of targeted mutation of a non-coding sequence in a region of interest in cellular chromatin in an isolated plant or mammalian cell containing a region of interest in cellular chromatin, the method comprising:
(a) selecting first and second nucleotide sequences in the region of interest;
(b) engineering first and second zinc finger binding domains to bind to the first and second nucleotide sequences;
(c) expressing a first fusion protein in the isolated cell, the first fusion protein comprising the first engineered zinc finger binding domain and a first FokI cleavage half-domain; and
(d) expressing a second fusion protein in the cell, the second fusion protein comprising the second zinc finger binding domain and a second FokI cleavage half-domain
wherein the first and second fusion proteins cleave cellular chromatin and mutate the non-coding sequence in the region of interest and further wherein one of the cleavage half-domains comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain.
2. The method of claim 1 , wherein cleavage occurs between the first and second nucleotide sequences.
3. The method of claim 1 , wherein non-coding sequences in the region of interest are deleted.
4. The method of claim 1 , wherein the mutation is selected from the group consisting of one or more nucleotide substitutions, one or more nucleotide deletions and one or more nucleotide insertions.
5. The method of claim 1 , wherein the non-coding sequence is a transcriptional regulatory sequence.
6. The method of claim 1 , wherein the first and second fusion proteins are expressed from different polynucleotides.
7. The method of claim 1 , wherein the first and second fusion proteins are expressed from a single polynucleotide.
8. A method of introducing an exogenous sequence into a non-coding region of interest of cellular chromatin of an isolated plant or mammalian cell, the method comprising,
mutating the non-coding sequence in the cell according to the method of claim 1 ; and
introducing a polynucleotide comprising the exogenous sequence into the cell, such that when the cellular chromatin is cleaved in the cell, the exogenous sequence is integrated into the non-coding region of interest.