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 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 editi...

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Autores principales:	Fiona M. Wilson, Richard J. Harrison
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	AMA1 CRISPR/Cas9 Codon optimisation Fusarium venenatum PolII promoter 5SrRNA Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/ec0d0cf46dc4492486f4f54cd49ab5b3
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spelling	oai:doaj.org-article:ec0d0cf46dc4492486f4f54cd49ab5b32021-11-21T12:28:41ZCRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS1210.1186/s40694-021-00121-82054-3085https://doaj.org/article/ec0d0cf46dc4492486f4f54cd49ab5b32021-11-01T00:00:00Zhttps://doi.org/10.1186/s40694-021-00121-8https://doaj.org/toc/2054-3085Abstract 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.Fiona M. WilsonRichard J. HarrisonBMCarticleAMA1CRISPR/Cas9Codon optimisationFusarium venenatumPolII promoter5SrRNABiotechnologyTP248.13-248.65ENFungal Biology and Biotechnology, Vol 8, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	AMA1 CRISPR/Cas9 Codon optimisation Fusarium venenatum PolII promoter 5SrRNA Biotechnology TP248.13-248.65
spellingShingle	AMA1 CRISPR/Cas9 Codon optimisation Fusarium venenatum PolII promoter 5SrRNA Biotechnology TP248.13-248.65 Fiona M. Wilson Richard J. Harrison CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12
description	Abstract 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.
format	article
author	Fiona M. Wilson Richard J. Harrison
author_facet	Fiona M. Wilson Richard J. Harrison
author_sort	Fiona M. Wilson
title	CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12
title_short	CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12
title_full	CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12
title_fullStr	CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12
title_full_unstemmed	CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12
title_sort	crispr/cas9 mediated editing of the quorn fungus fusarium venenatum a3/5 by transient expression of cas9 and sgrnas targeting endogenous marker gene pks12
publisher	BMC
publishDate	2021
url	https://doaj.org/article/ec0d0cf46dc4492486f4f54cd49ab5b3
work_keys_str_mv	AT fionamwilson crisprcas9mediatededitingofthequornfungusfusariumvenenatuma35bytransientexpressionofcas9andsgrnastargetingendogenousmarkergenepks12 AT richardjharrison crisprcas9mediatededitingofthequornfungusfusariumvenenatuma35bytransientexpressionofcas9andsgrnastargetingendogenousmarkergenepks12
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CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

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