Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum

Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8–1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.