The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first 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 identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.

CRISPR/Cas9 系统已成为丝状真菌靶基因敲除的重要工具。通过PCR或酶切法从大量转化体中鉴定突变体，既费力又费时。在这里，我们第一次研制出CRISPR / Cas9系统在米曲霉使用基于AMA1-自主复制质粒和Cas9所述的控制下，构巢曲霉gpdA启动子。通过基因组编辑技术，我们成功获得了米曲霉每个靶基因的突变. 然后，我们将目标基因的protospacer序列及其protospacer相邻基序（PAM）放在DsRed的起始密码子“ATG”后面，产生无功能的DsRed（nDsRed）报告基因，nDsRed报告基因可以在成功的有针对性的编辑。此外，该方法还通过靶向曲酸合成基因 kojA 应用，发现具有 DsRed 活性的转化体在kojA 中具有靶向突变。这些结果表明，nDsRed 可以作为一种强有力的工具来促进在米曲霉中识别由 CRISPR/Cas9 产生的突变体。