A versatile and efficient gene targeting system for Aspergillus nidulans

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for non-homologous end joining of DNA in double strand break repair. Deletion of nkuA (nkuA) greatly reduces the frequency of non-homologous integration of transforming DNA fragments leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA and the heterologous selectable markers make up a very efficient gene targeting system. In experiments, involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genome-wide gene targeting projects feasible.

Key Words: Aspergillus, Recombination, gene targeting, non-homologous end joining, transformation

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