PCR-based gene targeting in the filamentous fungus Ashbya gossypii

We have investigated a PCR-based approach for one-step gene targeting in the filamentous fungus Ashbya gossypii. Short guide sequences with 40-46 bp of homology to two sequences of a targeted gene, provided by PCR, were sufficient to mediate homologous recombination. The PCR products used for transformation were generated from the newly constructed chimeric selection marker GEN3. This consists of the open reading frame of the Escherichia coli kanR gene under the control of promoter and terminator sequences of the Saccharomyces cerevisiae TEF2 gene and allows selection of G418/geneticin-resistant transformants. Verification of gene targeting was performed either by PCR or by DNA hybridization analyses, and in all 18 cases tested, correct targeting was confirmed. This approach was used for the complete deletion of the open reading frame of the A. gossypii RHO4 gene for which a double-strand sequence was available as information source for the design of PCR primers. We also demonstrated successful partial deletion of four other ORFs using single-read sequences (SRS) as sole information for the design of targeting primers. A gossypii is the first filamentous fungus in which a PCR-based gene disruption technique has been established. Since short target guide sequences are sufficient to direct homologous integration into the A. gossypii genome it is not necessary to obtain and sequence large DNA fragments from a target locus to provide the long flanking homology regions usually required for efficient targeting of cloned disruption cassettes in filamentous fungi. Thus functional analysis of A. gossypii genes is already possible, based on single-pass sequence information.