Single and combinatorial gene inactivation in Aspergillus niger using selected as well as genome-wide gRNA library pools.

Aspergillus niger is a saprotroph, a pathogen, an endophyte, a food spoiler and an important cell factory. Only a minor fraction of its genes has been experimentally characterized. We here set up a CRISPR/Cas9 mutagenesis screen for functional gene analysis using co-transformation of a pool of gene editing plasmids that are maintained under selection pressure and that each contain a gRNA. First, a pool of gRNA vectors was introduced in A. niger targeting five genes with easy selectable phenotypes. Transformants were obtained with all possible single, double, triple, quadruple and quintuple gene inactivation phenotypes. Their genotypes were confirmed using the gRNA sequences in the transforming vector as barcodes. Next, a gRNA library was introduced in A. niger targeting > 9600 genes. Gene nsdC was identified as a sporulation gene using co-transformation conditions that favored uptake of one or two gRNA construct(s) from the genome-wide vector pool. Together, CRISPR/Cas9 vectors with a (genome-wide) pool of gRNAs can be used for functional analysis of genes in A. niger with phenotypes that are the result of the inactivation of a single or multiple genes.