Construction of an <i>Aspergillus oryzae</i> triple amylase deletion mutant as a chassis to evaluate industrially relevant amylases using multiplex CRISPR/Cas9 editing technology

Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities potential amylases, however, cannot be quantified directly from supernatant due to high background activity native α-amylase. This caused by gene products amyA, amyB, and amyC. In this study, in vitro CRISPR/Cas9 system was established A. delete these genes simultaneously. First, pyrG NSAR1 mutated exploiting NHEJ generate a counter-selection marker. Next, all amylase were deleted simultaneously co-transforming repair template carrying nidulans flanking sequences loci. rate obtained triple knock-outs 47%. We showed that knockouts do not retain any supernatant. used achieve sequence-specific knock-in target genes. intended incorporate single copy interest into desired host development screening methods. Therefore, integration cassette Fpi designed specifically amyB locus. site-specific plasmid 78%, with exceptional additional integrations. Integration frequency assessed via qPCR correlated activity. Hence, we could compare efficiency between two different signal peptides. summary, present strategy exploit mutation, multiplex knock-out, targeted expression oryzae. Our provides straightforward strain engineering paves way fungal systems.