Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei

Trichoderma reesei is a filamentous fungus that is used world-wide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement by mutagenesis and screening approaches. It is also desirable to specifically manipulate genes of the organism to further improve and modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation we have developed a simple transient method for silencing genes in T. reesei . Using gene specific siRNAs targeted to mus53 , we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we have demonstrated that transiently silencing DNA repair genes significantly improved homologous integration of DNA into a specific locus delivered in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration. Importance The previous solution for improving the targeted integration efficiency has been deleting non-homologous end joining DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to accumulation of spontaneous mutations. Our method of transiently silencing the NHEJ pathway genes allows for recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Further, our transient silencing method is a truly flexible approach capable of knocking down expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene function in T. reesei .