CRISPR/Cas9 genome editing for comparative genetic analysis related to soy sauce brewing in Aspergillus sojae...

Aspergillus sojae has traditionally been used in soy sauce brewing. Genetic modification techniques have been established in A. sojae, but it is difficult to apply them to various industrial strains. Although we have previously developed a CRISPR/Cpf1 system for genetic modification of A. sojae, another genome editing system was required for versatile modification. In addition, repetitive genetic modification using the CRISPR system has not been established in A. sojae. In this study, we demonstrated mutagenesis, gene deletion/integration, and large deletion of a chromosomal region in A. sojae using the CRISPR/Cas9 system. We also successfully performed repetitive genetic modification using a method that involved forced recycling of genome-editing plasmids. Moreover, we demonstrated that the effects of genetic modification related to soy sauce brewing differed among A. sojae industrial strains. These results showed that our technique of using the CRISPR/Cas9 system is a powerful tool for genetic modification in A. sojae.