Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.
Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.
Bibikova
M
M
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Carroll
D
D
Segal
D J
DJ
Trautman
J K
JK
Smith
J
J
Kim
Y G
YG
Chandrasegaran
S
S
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D008845:Microinjections; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014982:Xenopus laevis; D016335:Zinc Fingers
D011993:Recombinant Fusion Proteins; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific