CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea

• Published in: PLoS pathogens Vol. 16; no. 8; p. e1008326
• Main Authors: Leisen, Thomas, Bietz, Fabian, Werner, Janina, Wegner, Alex, Schaffrath, Ulrich, Scheuring, David, Willmund, Felix, Mosbach, Andreas, Scalliet, Gabriel, Hahn, Matthias, Thomma, Bart, Bolton, Melvin
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 17.08.2020; Public Library of Science (PLoS)
• Subjects: Amino acids; Antigens; Apoptosis; Biology; Biology and Life Sciences; Botrytis; Botrytis cinerea; Codons; CRISPR; CRISPR-Cas technology; Editing; Efficiency; Engineering and Technology; Funding; Fungi; Fungicides; Gene sequencing; Genes; Genetic aspects; Genetic diversity; Genetic engineering; Genome editing; Genomes; Health aspects; Kinases; Markers; Methods; Mutation; Pathogens; Pesticides; Physiology; Plasmids; Proteins; Protoplasts; Random mutagenesis; Ribonucleoproteins; Succinate dehydrogenase; Telomeres; Germany; Aachen Germany; Switzerland
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1008326

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.