Establishment of an Efficient CRISPR-Cas9-Mediated Gene Disruption System in the Lichen-Forming Fungus Umbilicaria muhlenbergii.

Lichen-forming fungi establish intimate symbiotic associations with photosynthetic partners and play important roles in diverse ecosystems, but functional genetic studies in these organisms remain limited by the lack of efficient genome-editing tools. In this study, we established an efficient CRISPR-Cas9-mediated gene disruption system in Umbilicaria muhlenbergii. Using this system, we achieved the targeted disruption of six candidate transcription factors with a high replacement efficiency of up to 65.0%. No off-target mutations were detected in any of the three independent mutants examined for each target gene. Preliminary phenotypic characterization of the resulting mutants revealed that disruption of UmSOM1 markedly impaired fungal growth, induced pseudohyphal development, and altered colony morphology and pigmentation. Compared with conventional homologous recombination, the CRISPR-Cas9 system substantially improved gene disruption efficiency, thereby overcoming a major limitation in the genetic manipulation of lichen-forming fungi. This system provides a robust platform for functional genomic studies and will accelerate investigations into the molecular mechanisms underlying fungal-algal symbiosis and morphological transitions in lichen-forming fungi.