Targeted gene insertion or replacement is a promising genome editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum , targeted gene insertion and replacement remains a serious challenge due to the low efficiency of homologous recombination (HR) in these species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the non-homologous end joining pathway (NHEJ) at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma , we first inactivated the NHEJ pathway via disruption of the Ku70 gene ( ku70 ) using a dual sgRNA-directed gene deletion method. Disruption of the ku70 significantly decreased NHEJ activity in G. lucidum . Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of a targeted gene insertion and replacement when target DNA orotidine 5’-monophosphate decarboxylase gene ( ura3 ) with 1.5 kb 5’ and 3’ homologous flanking sequences were used as a donor template, compared to 3.3% and 0% for a control strain (Cas9 strain) at these targeted sites, respectively. Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum . Importance Functional genomic studies have been hindered in Ganoderma by the absence of adequate genome engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum , targeted gene insertion and replacement has remained a serious challenge due to the low efficiency of homologous recombination in these species, although such precise genome modifications including site mutations, site-specific integrations and allele or promoter replacements would be incredibly valuable. In this work, we inactivated the non-homologous end joining repair mechanism in G. lucidum by disrupting the ku70 using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70 -disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

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