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Molecular and Cellular Biology, January 2001, p. 289-297, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.289-297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases

Marina Bibikova,1 Dana Carroll,1,* David J. Segal,1,dagger Jonathan K. Trautman,1 Jeff Smith,2,3 Yang-Gyun Kim,2,Dagger and Srinivasan Chandrasegaran2,*

Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132,1 and Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health,2 and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine,3 Baltimore, Maryland 21205

Received 28 August 2000/Returned for modification 2 October 2000/Accepted 5 October 2000

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.

* Corresponding author. Mailing address for Dana Carroll: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132. Phone: (801) 581-5977. Fax: (801) 581-7959. E-mail: carroll{at}path.utah.edu. Mailing address for Srinivasan Chandrasegaran: Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 614-2289. Fax: (410) 955-0617. E-mail: chandra{at}welchlink.welch.jhu.edu.

dagger Present address: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

Dagger Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Molecular and Cellular Biology, January 2001, p. 289-297, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.289-297.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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