A highly efficient identification of mutants generated by CRISPR/Cas9 using the non‑functional DsRed assisted selection in Aspergillus oryzae

Abstract

The CRISPR/Cas9 system has become a great tool for target gene knock-out in lamentous fungi. It is laborious and time-consuming that identi cation mutants from a large number of transformants through PCR or enzyme-cut method. Here, we rst developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae. Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon "ATG" of DsRed, yielding the non-functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing.Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identi cation of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.

A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

“…The U6 terminator and sgRNA sequence were amplified from the vector pPTRII-Cas9-AoGld3 with the primers TU6-Aokap4-F and TU6-R, yielding the fragment Aokap4-TU6. The two fragments above were fused by the primers PU6-F and TU6-R, and inserted into the SmaI site of the pPTRII-Cas9 vector [ 27 ], yielding the plasmid pPTRII-Cas9-Aokap4. The constructed plasmid was transformed into the A. oryzae 3.042 strain.…”

“…The constructed plasmid pPTRII-Cas9-Aokap4 was transformed into a kojT -deletion strain generated previously by us [ 27 ]. The transformants were subcultured on the CD medium containing pyrithiamine and verified via sequencing.…”

Aspergillus oryzae accelerates the conversion of ergosterol to ergosterol peroxide by efficiently utilizing cholesterol

“…Cholesterol CD medium was made by the Czapek-Dox (CD) medium sterilized at 115°C for 15 min ( Li et al, 2021 ). Depending on the concentration of cholesterol medium to be made, the appropriate amount of cholesterol (99%) was sterilized using ultraviolet radiation for 30–40 min, added to the above sterile CD medium and shaken well.…”

Identification and characterization of Nramp transporter AoNramp1 in Aspergillus oryzae

“…The synthetic sgRNA-TU6 was used as the template to amplify the DNA fragment AoNramp1-sgRNA-TU6 with the primer sets, TU6-R and AoNramp1-TU6-F attached with the target sequence for AoNramp1. The above-mentioned two fragments containing the target sequence for AoNramp1, PU6-AoNramp1 and AoNramp1-sgRNA-TU6, were fused by overlapping PCR, then inserted into the SmaI-digested CRISPR/Cas9 vector pPTRII-Cas9 containing the Cas9 under the control of the A. nidulans gpdA promoter (PgpdA) and the pyrithiamine resistance marker ptrA (Li et al 2021), yielding the plasmid pPTRII-Cas9-AoNramp1. The resulting plasmid was introduced into 3.042 strain by the PEG-protoplast method.…”