High-throughput construction of gene deletion cassettes for generation of Neurospora crassa knockout strains.

The availability of complete genome sequences for a number of biologically important fungi has become an important resource for fungal research communities. However, the functions of many open reading frames (ORFs) identified through annotation of whole genome sequences have yet to be determined. The disruption of ORFs is a practical method for loss-of-function gene analyses in fungi that are amenable to transformation.

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.

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.