Methods and compositions for targeted single-stranded cleavage and targeted integration
First Claim
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1. A method of modifying a genomic sequence in a cell, the method comprising introducing a polynucleotide encoding an artificial nuclease into the cell, the 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;
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Abstract
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.
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12 Claims
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1. A method of modifying a genomic sequence in a cell, the method comprising introducing a polynucleotide encoding an artificial nuclease into the cell, the 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 genomic sequence is modified. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of detecting a nucleic acid comprising:
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generating a single-stranded break (nick) in a double-stranded DNA of a sample using 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 DNA and the nuclease induces a single-stranded break in the double-stranded DNA; amplifying the nicked DNA; and detecting the amplified DNA, thereby detecting the nucleic acid. - View Dependent Claims (9, 10, 11, 12)
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Specification