Selection is required for efficient Cas9-mediated genome editing in Fusarium graminearum

Abstract

Genome engineering using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nucleases, such as Cas9 (CRISPR-associated protein 9), are revolutionising molecular biology. In this study, we established a Cas9-based genome editing system in Fusarium graminearum, a highly destructive fungal pathogen of cereal crops. Although the molecular toolkit of F. graminearum is well developed compared to other fungi, Cas9-mediated engineering offers a number of potential benefits, such as the ability to create marker free mutants in this species. Here we have used a codon-optimised Cas9 nuclease and dual ribozyme-based expression of a single guide RNA (sgRNA) to induce mutations. Cas9-mediated mutations were identified through a fungicide resistance-based phenotypic screen, which selects for null mutations in the FgOs1 gene encoding an osmosensor histidine kinase. In the absence of selection, however, mutations were identified at very low frequency. Examination of the mutant alleles identified suggests that, a microhomology-mediated end joining (MMEJ) DNA repair pathway is likely to be the predominant process involved in erroneous repairing of Cas9-induced double-stranded breaks in F. graminearum.