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To establish a system that greatly increases the gene-targeting frequency in Aspergillus parasiticus.The ku70 gene, a gene of the nonhomologous end-joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O-methylsterigmatocystin (OMST). The NHEJ-deficient strain, RHΔku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA-topoisomerase I complex inhibitor, camptothecin, and the DNA-damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA-targeting frequency in RHΔku70 reached 96%.The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ-deficient RHΔku70, easy creation of homologous ends for integration, and the ptr-based selection form a highly efficient gene-targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies.The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.