Systematic Profiling of ssODN Determinants Establishes Optimized LbCas12a-Mediated HDR for Precise Genome Editing in Flammulina filiformis

Although gene editing technologies have advanced considerably, research on gene editing in Flammulina filiformis remains in its infancy, and a systematic evaluation of how ssODN donor properties affect HDR efficiency in this species is still lacking. To enable precise genome editing in the edible mushroom F. filiformis, we established an LbCas12a ribonucleoprotein (RNP)-mediated homology-directed repair (HDR) platform in protoplasts and systematically evaluated key ssODN donor design parameters: PAM-site mutation, homology arm distance, RAD51-preferred sequences (RPSs), truncated Cas target sequences (tCTSs), the RS-1 enhancer, and thermodynamic stability (ΔG). A PAM-disrupting mutation greatly increased HDR efficiency, up to 58.5%. Extending the left homology arm 174 bp upstream of the break still yielded 20.2% HDR, expanding the tolerable distance. RPSs showed context-dependent effects: enhancing HDR up to 287.8% in some designs but reducing it by 61.9% in another. Both tCTS and RS-1 consistently suppressed HDR. No strong linear correlation between ΔG and HDR efficiency was observed. Collectively, from experiments conducted at a single genomic locus, our results not only establish a versatile precision-editing platform for F. filiformis through optimized ssODN design, but also offer tentative and transferable insights into the HDR-mediated genome editing of other heterokaryotic basidiomycetes facing similar genetic operation challenges.