- 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 RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and
other fungi.
