CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

Abstract

BackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn). ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.