ArticlePLoS pathogens2020
CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.
Article in PLoS pathogens, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 55 papers.
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55 citing papers in PubMed, 109 citations in OpenAlex.
- Resistance of Botrytis cinerea to anilinopyrimidine fungicides: A novel ARMS-PCR method for the detection of Bcpos5 mutations and characterization of resistance using CRISPR/Cas9 editing.Pest management science · 2026Article
- Metabolic reprogramming of abscisic acid-producing strainSynthetic and systems biotechnology · 2026Article
- Plant-associated fungi co-opt ancient antimicrobials for host manipulation.Science advances · 2026Article
- Optimization of CRISPR/Cas9-based multi-gene editing system in Aspergillus oryzae and its application in α-amyrin biosynthesis.World journal of microbiology & biotechnology · 2026Article
- A High-Efficiency CRISPR-Cas9 Ribonucleoprotein Genome Editing System inJournal of fungi (Basel, Switzerland) · 2026Article
- Loss of virulence ofmBio · 2026Article
- Exploring salicylic acid biosynthesis in Trichoderma spp. using an enhanced transformation approach.Fungal biology and biotechnology · 2026Article
- Advances in molecular tools for elucidating nucleic acid biology in fungal pathogens.microLife · 2026Review
- Generation of Seamless Point Mutations with Cas9 RNP and pTel-hygR Plasmid in Aspergillus fumigatus.Methods in molecular biology (Clifton, N.J.) · 2026Article
- DNA Aptamers Targeting BcSOD1: A Novel Strategy for Controlling Botrytis cinerea in Sustainable Agriculture.Plant biotechnology journal · 2025Article
- Saprotrophic Arachnopeziza Species as New Resources to Study the Obligate Biotrophic Lifestyle of Powdery Mildew Fungi.Molecular ecology resources · 2025Article
- Current status and future trends of eco-friendly management of postharvest fungal decays in tomato fruit.NPJ science of food · 2025Review
- Enhancing abscisic acid production inSynthetic and systems biotechnology · 2025Article
- Genetic and Genomic Analysis IdentifiesInternational journal of molecular sciences · 2025Article
- Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species.Microbiology spectrum · 2025Article
- Unravelling the interplay of nitrogen nutrition and the Botrytis cinerea pectin lyase BcPNL1 in modulating Arabidopsis thaliana susceptibility.Communications biology · 2025Article
- Editorial: Molecular interactions and control strategies forFrontiers in plant science · 2025Article
- Simply cut out - Combining CRISPR/Cas9 RNPs and transiently selected telomere vectors for marker free-gene deletion inFrontiers in genome editing · 2025Article
- Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.Journal of fungi (Basel, Switzerland) · 2024Review
- Dual RNA-seq reveals distinct families of co-regulated and structurally conserved effectors in Botrytis cinerea infection of Arabidopsis thaliana.BMC biology · 2024Article
Corrections and comments
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Authors and funding
10 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.
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