Targeted deletion of cellular DNA sequences
Disclosed herein are methods and compositions for targeted deletion of double-stranded DNA. 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 deletion include introduction of such fusion proteins, or polynucleotides encoding same, into a cell such that two targeted cleavage events occur. Subsequent cellular repair mechanisms result in deletion of sequences between the two cleavage sites.
1. A method for deleting at least 100 nucleotide pairs in a region of interest in endogenous double-stranded DNA in a plant or mammalian cell, the method comprising:
expressing first, second, third and fourth fusion proteins in the cell, each of the fusion proteins comprising:
(i) a zinc finger DNA-binding domain that binds to a target site in the endogenous DNA, and
(ii) a cleavage half-domain;
further wherein:
(a) the first and second fusion proteins bind to first and second target sites respectively, wherein a first cleavage site lies between the first and second target sites, wherein the first and second target sites, each target site comprising at least 3 target subsites, and
(b) the third and fourth fusion proteins bind to third and fourth target sites respectively, wherein a second cleavage site lies between the third and fourth target sites and wherein the first and second cleavage sites are within different sequences;
such that the first and second fusion proteins cleave the DNA at the first cleavage site, the third and fourth fusion proteins cleave the DNA at the second cleavage site, and DNA ends are rejoined such that at least 100 endogenous nucleotide pairs between the first and second cleavage sites are deleted and at least two of the target sites are maintained in the endogenous double-stranded DNA following deletion of the at least 100 endogenous nucleotide pairs.
2. The method of claim 1 , wherein the first and second cleavage sites are on the same chromosome.
3. The method of claim 1 , wherein the near edges of the first and second target sites are separated by between 4 and 6 nucleotide pairs.
4. The method of claim 1 , wherein the near edges of the third and fourth target sites are separated by between 4 and 6 nucleotide pairs.
5. The method of claim 1 , wherein at least 400 nucleotide pairs between the first and second cleavage sites are deleted.
6. The method of claim 5 , wherein at least 1000 nucleotide pairs between the first and second cleavage sites are deleted.
7. The method of claim 1 , wherein the cell is a primary cell.
8. The method of claim 7 , wherein the cell is a T-cell.
9. The method of claim 1 , wherein the first and second cleavage sites are separated by more than 100 nucleotide pairs.
10. The method of claim 1 , wherein the target site is in a CCR5 gene, β2 microglobulin gene or CTLA4 gene.