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

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dc.citation.issue1
dc.citation.volume126
dc.contributor.authorRocafort M
dc.contributor.authorArshed S
dc.contributor.authorHudson D
dc.contributor.authorSidhu JS
dc.contributor.authorBowen JK
dc.contributor.authorPlummer KM
dc.contributor.authorBradshaw RE
dc.contributor.authorJohnson RD
dc.contributor.authorJohnson LJ
dc.contributor.authorMesarich CH
dc.contributor.editorBrown NA
dc.coverage.spatialNetherlands
dc.date.accessioned2023-12-15T01:34:26Z
dc.date.accessioned2024-07-25T06:36:35Z
dc.date.available2021-10-09
dc.date.available2023-12-15T01:34:26Z
dc.date.available2024-07-25T06:36:35Z
dc.date.issued2022-01
dc.description.abstractApple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) 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 THN 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 THN 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 V. inaequalis. 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 V. inaequalis, 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.
dc.description.confidentialfalse
dc.edition.editionJanuary 2022
dc.format.pagination35-46
dc.identifier.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/34930557
dc.identifier.citationRocafort M, Arshed S, Hudson D, Sidhu JS, Bowen JK, Plummer KM, Bradshaw RE, Johnson RD, Johnson LJ, Mesarich CH. (2022). CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis.. Fungal Biol. 126. 1. (pp. 35-46).
dc.identifier.doi10.1016/j.funbio.2021.10.001
dc.identifier.eissn1878-6162
dc.identifier.elements-typejournal-article
dc.identifier.issn1878-6146
dc.identifier.piiS1878-6146(21)00132-X
dc.identifier.urihttps://mro.massey.ac.nz/handle/10179/70529
dc.languageeng
dc.publisherElsevier BV on behalf of the British Mycological Society
dc.publisher.urihttps://www.sciencedirect.com/science/article/pii/S187861462100132X
dc.relation.isPartOfFungal Biol
dc.rights(c) 2022 The Author/s
dc.rightsCC BY-NC-ND 4.0
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectApple scab
dc.subjectCRISPR-Cas9 gene editing
dc.subjectFungus
dc.subjectHigh-resolution melting
dc.subjectMelanin biosynthesis pathway
dc.subjectTrihydroxynaphthalene reductase gene
dc.subjectVenturia inaequalis
dc.subjectAscomycota
dc.subjectCRISPR-Cas Systems
dc.subjectFungal Genus Venturia
dc.subjectGene Editing
dc.subjectMalus
dc.subjectPlant Diseases
dc.titleCRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis
dc.typeJournal article
pubs.elements-id449150
pubs.organisational-groupOther
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