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Background  Gene 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 various 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™).    Results  We 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. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing  cas9  codon-optimised for  F. venenatum .    Conclusions  Using 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  for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.    Supplementary Information  The online version contains supplementary material available at 10.1186/s40694-021-00121-8.

fungal biology and biotechnology

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