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BIOLOGICAL SCIENCES / GENETICS
A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

Hildur V. Colot^*,, Gyungsoon Park^,, Gloria E. Turner, Carol Ringelberg^*, Christopher M. Crew^,¶, Liubov Litvinkova, Richard L. Weiss, Katherine A. Borkovich, and Jay C. Dunlap^*,||

*Department of Genetics, Dartmouth Medical School, HB7400, Hanover, NH 03755; Department of Plant Pathology, University of California, Riverside, CA 92521; and Department of Chemistry and Biochemistry, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095

Edited by David D. Perkins, Stanford University, Stanford, CA, and approved March 29, 2006 (received for review February 21, 2006)

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

filamentous fungi | functional genomics | knockouts | recombinational cloning

H.V.C. and G.P. contributed equally to this work.

^¶Present address: School of Psychology, Georgia Institute of Technology, 654 Cherry Street, Atlanta, GA 30332-0170.

Author contributions: H.V.C., G.P., G.E.T., K.A.B., and J.C.D. designed research; H.V.C., G.P., G.E.T., C.R., C.M.C., and L.L. performed research; H.V.C., G.P., G.E.T., C.R., C.M.C., R.L.W., K.A.B., and J.C.D. contributed new reagents/analytic tools; H.V.C., G.P., G.E.T., and K.A.B. analyzed data; and H.V.C., G.P., G.E.T., K.A.B., and J.C.D. wrote the paper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

^||To whom correspondence should be addressed. E-mail: jay.c.dunlap{at}dartmouth.edu

© 2006 by The National Academy of Sciences of the USA

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