Use of chimeric nucleases to stimulate gene targeting
Gene targeting is a technique to introduce genetic change into one or more specific locations in the genome of a cell. For example, gene targeting can introduce genetic change by modifying, repairing, attenuating or inactivating a target gene or other chromosomal DNA. In one aspect, this disclosure relates to methods and compositions for gene targeting with high efficiency in a cell. This disclosure also relates to methods of treating or preventing a genetic disease in an individual in need thereof. Further disclosed are chimeric nucleases and vectors encoding chimeric nucleases.
1. A transfection system comprising:
(a) a nucleic acid encoding first and second chimeric nucleases, each chimeric nuclease comprising:
(i) a zinc finger DNA binding domain that binds to a target site in an endogenous beta globin or CD8 mammalian gene, wherein the target site comprises SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 38, nucleotides 19 to 30 of SEQ ID NO: 51 or nucleotides 6 to 14 of SEQ ID NO: 52;
(ii) a FokI cleavage domain; and
(iii) a nuclear localization signal,
wherein the first and second chimeric nucleases form a dimer and cleave the endogenous mammalian gene at the cleavage site; and
(b) a nucleic acid comprising a repair substrate that comprises:
(i) a nucleic acid sequence that is substantially identical to a region flanking a target sequence in chromosomal DNA; and
(ii) a nucleic acid sequence which is integrated into the cleavage site upon cleavage by the chimeric nucleases and recombination between the repair substrate and the target sequence.
2. An isolated mammalian cell comprising:
(a) first and second chimeric nucleases, each nuclease comprising a zinc finger DNA-binding domain that binds to a site in an endogenous beta globin CD8 mammalian gene and a FokI cleavage domain, wherein the site comprises SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:38, nucleotides 19 to 30 of SEQ ID NO:51 or nucleotides 6 to 14 of SEQ ID NO:52,
wherein the first and second chimeric nucleases form a dimer and cleave the endogenous mammalian gene at a cleavage site; and
(b) a repair substrate comprising
(i) a nucleic acid sequence that is substantially identical to a region flanking a target sequence in endogenous chromosomal DNA; and
(ii) a nucleic acid sequence which is integrated into the cleavage site upon cleavage by the chimeric nucleases and recombination between the repair substrate and the target sequence.