Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae

Abstract

We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

Highly efficient gene targeting in <i>Aspergillus oryzae</i> industrial strains under <i>ligD</i> mutation introduced by genome editing: Strain-specific differences in the effects of deleting EcdR, the negative regulator of sclerotia formation

“…The plasmid for ligD gene mutation by the CRISPR/Cas9 system was constructed as follows; the U6 promoter with the ligD target sequence was amplified from the plasmid pUNAFNC9gwA1 (Katayama et al, 2016) using primers pUNA+U6p-F and U6p-ligD-R1. The U6 terminator and sgRNA sequence were amplified from pUNAFNC9gwA1 using the primers ligD-U6t-F1 and pUNA+U6p+gRNA+U6t-R3.…”

“…The U6 terminator and sgRNA sequence were amplified from pUNAFNC9gwA1 using the primers ligD-U6t-F1 and pUNA+U6p+gRNA+U6t-R3. These two fragments were fused using the primers pUNA+U6p-F and pUNA+U6p+gRNA+U6t-R3, and inserted into the XbaI site of the plasmid pUNAFNcas9 (Katayama et al, 2016), and the obtained plasmid pUNAFNcas9lD was introduced into the niaD mutants. Transformants were selected on the CD medium, and mutations in the ligD gene were analyzed by nucleotide sequencing.…”

“…When the DNA strand cleaved by Cas9 is repaired, mutation is introduced at the target locus. The functionality of the CRISPR/Cas9 system as a genome editing method has been reported in filamentous fungi (Arazoe et al, 2015;Fuller et al, 2015;Katayama et al, 2016;Liu et al, 2015;Nødvig et al, 2015). We recently developed the CRISPR/Cas9 system as a genome editing method in A. oryzae and demonstrated that the system can be applied to industrial strains (Katayama et al, 2016).…”

“…The functionality of the CRISPR/Cas9 system as a genome editing method has been reported in filamentous fungi (Arazoe et al, 2015;Fuller et al, 2015;Katayama et al, 2016;Liu et al, 2015;Nødvig et al, 2015). We recently developed the CRISPR/Cas9 system as a genome editing method in A. oryzae and demonstrated that the system can be applied to industrial strains (Katayama et al, 2016). However, the efficiency of mutagenesis using the CRISPR/Cas9 system was 10~20% (Katayama et al, 2016), which is lower than the gene targeting efficiencies in the deletion of ku70 and ligD (Maruyama and Kitamoto, 2008;Mizutani et al, 2008;Takahashi et al, 2006).…”

“…We recently developed the CRISPR/Cas9 system as a genome editing method in A. oryzae and demonstrated that the system can be applied to industrial strains (Katayama et al, 2016). However, the efficiency of mutagenesis using the CRISPR/Cas9 system was 10~20% (Katayama et al, 2016), which is lower than the gene targeting efficiencies in the deletion of ku70 and ligD (Maruyama and Kitamoto, 2008;Mizutani et al, 2008;Takahashi et al, 2006). Therefore, mutation of ku70 or ligD genes using the CRISPR/Cas9 system would be effective for efficient gene targeting.…”