Abstract:Flammulina filiformis has high nutritional and medicinal value. The CRISPR-Cas9 genome editing technology has been rapidly developed in recent years, with some successful applications in edible and medicinal fungus. However, its editing efficiency is comparatively low. The author targeted the pyrG gene for mutation, designing and synthesizing guide RNA (gRNA) in vitro. The gRNA was assembled with the SpCas9 protein to form ribonucleoprotein (RNP) complexes, which were introduced into the protoplasts of Flammulina filiformis using the PEG method, while the transformation parameters were optimized. The results demonstrated that computationally predicted high-scoring gRNA might not always exhibit activity and needed to be assembled with SpCas9 protein in vitro for cleavage verification of the target gene. Active RNP complexes specifically edited the target gene upon transformation into Flammulina filiformis protoplasts, whereas inactive complexes failed to induce genetic modifications. Adding Triton X-100 to the transformation system significantly enhanced RNP uptake into protoplasts. Sanger sequencing of mutant strains revealed small fragment insertions at the target site, indicating that genome modifications occurred primarily through the non-homologous end joining (NHEJ) pathway. The author successfully established an in vitro CRISPR-Cas9 genome editing technology in Flammulina filiformis, achieving an editing efficiency of 60%. Meanwhile, this study provides a theoretical basis and technical support for the breeding of new varieties of Flammulina filiformis with desirable traits, as well as other edible fungi.
