Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes.

Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.