CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, <i>Venturia inaequalis</i>

<p dir="ltr">Apple scab, caused by the fungal pathogen <i>Venturia inaequalis</i>, is the most economically important disease of apple (<i>Malus x domestica</i>) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of <i>V. inaequalis</i> is required. A major bottleneck for the genetic characterization of <i>V. inaequalis</i> is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in <i>V. inaequalis</i> have yet been published. Using the melanin biosynthesis pathway gene <i>trihydroxynaphthalene</i> reductase (<i>THN</i>) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the <i>THN</i> gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six <i>THN</i> mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of <i>V. inaequalis</i>. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in <i>V. inaequalis</i>, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.</p>