Targeted deletion of cellular DNA sequences
First Claim
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1. A method for deleting sequences in a region of interest in double-stranded DNA of genomic cellular chromatin in a 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 DNA, and(ii) a FokI cleavage half-domain,wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain; and
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, 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;
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 sequences between the first and second cleavage sites are deleted.
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Abstract
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.
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Citations
12 Claims
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1. A method for deleting sequences in a region of interest in double-stranded DNA of genomic cellular chromatin in a cell, the method comprising:
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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 DNA, and (ii) a FokI cleavage half-domain, wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain; and 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, 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; 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 sequences between the first and second cleavage sites are deleted. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for targeted replacement of a genomic sequence, the method comprising:
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(a) 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 DNA, and (ii) a FokI cleavage half-domain; wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain wherein 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, and 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; such that the first and second fusion proteins cleave the DNA at the first cleavage site, and the third and fourth fusion proteins cleave the DNA at the second cleavage site; and (b) contacting the cell with a donor polynucleotide, wherein the donor polynucleotide comprises; (i) sequences homologous to genomic sequences flanking the first and second cleavage sites; and (ii) sequences homologous but non-identical to genomic sequences between the first and second cleavage sites; whereby genomic sequences between the first and second cleavage sites are replaced by the homologous but non-identical sequences of the donor polynucleotide. - View Dependent Claims (9, 10, 11)
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12. A method for targeted replacement of a genomic sequence, the method comprising:
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(a) 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 DNA, and (ii) a FokI cleavage half-domain, wherein the cleavage half-domain of at least one of the fusion proteins comprises an alteration in amino acid 490 of the wild-type dimerization interface of the cleavage half-domain; wherein 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, and 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; such that the first and second fusion proteins cleave the DNA at the first cleavage site, and the third and fourth fusion proteins cleave the DNA at the second cleavage site; and (b) contacting the cell with a donor polynucleotide, wherein the donor polynucleotide comprises; (i) sequences homologous to genomic sequences flanking the first and second cleavage sites; and (ii) sequences that are non-homologous to genomic sequences between the first and second cleavage sites; whereby genomic sequences between the first and second cleavage sites are replaced by the non-homologous sequences of the donor polynucleotide.
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Specification