Development of CRISPR-Cas systems for genome and transcriptome editing in fusarium circinatum

Abstract

Fusarium circinatum, the causal agent of pine pitch canker disease, poses a significant threat to the health of Pinus species worldwide. Infection by this pathogen results in resinous canker formation in adult pine trees, and root and collar rot in seedlings, resulting in a severe mortality rate. While much is known about this pathogen, there is a lack in the understanding of the underlying virulence mechanisms. To date very few genes have been functionally characterized and shown to play a role in F. circinatum pathogenicity which is likely due a lack of efficient genome and transcriptome editing systems. For this purpose, we established a CRISPR-Cas9 and CRISPR-Cas13 system to enable editing at the DNA and RNA levels, respectively. The CRISPR-Cas9-mediated gene knockout system was established in F. circinatum to functionally characterize a putative pathogenicity gene. The pgs gene was found to play a role in pathogenicity, growth and sporulation when comparing various knockout isolates to their respective wild type isolates in various phenotypic assays. The CRISPR-Cas13 system was developed and established in F. circinatum as a proof of concept for the in vivo use of the RNA-targeting system in filamentous fungi. The system was capable of targeting GFP transcripts in GFP-expressing isolates resulting in a decrease in GFP fluorescence. This research expands the molecular toolbox available for functional characterization, RNA regulation, and editing, not only in F. circinatum, but in related species as well.