CRISPR/Cas9 improves targeted knock-in efficiency in Aspergillus oryzae.

Aspergillus oryzae is an important fungus in food and industrial enzyme production. In A. oryzae, targeted knock-in transformation is primarily limited to homologous recombination (HR)-based systems, in which non-homologous end-joining (NHEJ)-disruptant hosts are required. However, preparation of hosts and transformation templates for such systems is laborious, in addition to other disadvantages. In the present study, we examined alternative targeted knock-in mediated by CRISPR/Cas9, in which a microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA) repair system was employed. This approach enabled the efficient development of targeted knock-in transformants without host preparation using only a short homology template. We conclude that this new method could be applied to facilitate the transformation of A. oryzae, and will make it easier to acquire targeted knock-in transformants, especially from industrially important non-model strains.