Constructing pyrG marker by CRISPR/Cas facilities the highly-efficient precise genome editing on industrial Aspergillus niger strain.

To prevent the unique difficulty of hygromycin-based gene editing on industrial A. niger strain and increase the working efficiency, the local pyrG marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired pyrG auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δku70 and Δku80 respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and amdS counter-selection repair template were used to obtain ERG3^Tyr185 mutation. A highly-efficient precise strategy was established for A. niger through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.