Cas9 Ribonucleoproteins (RNPs) for Gene Deletion

Genetic manipulations in pathogenic microorganisms, like Aspergillus fumigatus, allow us to study the function of genes in pathogenicity or other important traits. Classic genetic engineering tools in filamentous fungi rely on DNA fragment sub-cloning or fusion PCR to build a gene deletion cassette containing extended flanking regions of homology to promote efficient recombination. However, the introduction of CRISPR technology into fungal genetics has greatly simplified and accelerated the genome editing process. Here, we describe a simple and universal, one-step CRISPR-Cas9-mediated genetic tool employing repair templates containing microhomology regions. This gene targeting system displays high homologous recombination efficiency rates and can be easily implemented across different genetic backgrounds.