Gene editing of Ganoderma lucidum using CRISPR/Cas9 ribonucleoprotein complex

Gene editing of Ganoderma lucidum using CRISPR/Cas9 ribonucleoprotein complex

Hyerang Eom1, Min-Seek Kim2 and Hyeon-Su Ro1*

1Department of Bio&Medical Bigdata (BK21plus) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Korea
2Mushroom Research Division, National Institute of Horticultural & Herbal Science, RDA, Eumseong, Chungbuk, Korea
*Email: rohyeon@gnu.ac.kr

Gene editing presents a promising alternative to conventional breeding when it comes to developing new strains of mushrooms. However, the current method often involves using Cas9-plasmid DNA raising concerns about the creation of genetically modified organisms. In this research, we accomplished the editing of the pyrG gene in Ganoderma lucidum by utilizing a preassembled complex of Cas9 and guide RNA (gRNA). This complex primarily induced a break in the DNA at the fourth position just before the protospacer adjacent motif. Among the 66 edited organisms, 42 displayed deletions ranging from a single missing base to large deletions of up to 674 base pairs. Notably, 30 out of these deletions were single-base deletions. Interestingly, the remaining 24 organisms had inserted sequences of various sizes at the site of the DNA break. These sequences originated from fragments of the host’s mitochondrial DNA, the chromosomal DNA of E. coli, and the DNA of the Cas9 expression vector. The last two sources were likely contaminants that were not eliminated during the purification process of the Cas9 protein. Despite this unexpected outcome, the study underscored that editing genes in G. lucidum using the Cas9-gRNA complex is achievable with efficiency comparable to the plasmid-mediated editing system. [Supported by New Breeding Technologies Development Program Project No. RS-2022-RD010233, RDA, Korea]