Recruitment of tRNA PolIII promoters in CRISPR/Cas9 system to edit genome of Aspergillus niger and the application in strain development

As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in few organisms of filamentous fungi that greatly improved the efficiency of gene alteration. However, the delivery of the guide RNA (gRNA) has been so far the bottleneck of performing CRISPR mutagenesis on Aspergillus species. Here we report a modification of gRNA expression driven by a series of endogenous tRNA promoters, which include the tRNA gene plus preceding 100-basepair of upstream sequence. Cotransformation of Cas9-expressing plasmid with gRNA linear DNA demonstrated that 97% of 37 tRNA promoters are able to produce mature gRNA for Cas9 targeting in Aspergillus niger. When gRNA(s) and Cas9 were expressed in a single extrachromosomal plasmid, the efficiency of single gene disruption was achieved as high as 98%, and the duplex targeting efficiency was around 68%. Along with a homologous recombination donor, our CRISPR/Cas9 system resulted in 94% efficiency of gene knock-in in the kusA^- strain, which was 100% of targeting integration. Relied on robust and reliable regulatory strength of tRNA promoter, our results provide a wide avenue of genome editing in A. niger and in other filamentous fungi. In addition, using tRNAs as polymerase III promoter to lead gRNA expression in the CRISPR/Cas9 system can be easily extended to other organisms than fungi.