Eom Hyerang, Choi Yeon-Jae, Nandre Rutuja, Kim Minseek, Oh Youn-Lee, Kim Sinil, Nakazawa Takehito, Honda Yoichi, Ro Hyeon-Su
Department of BioMedical Bigdata (BK21) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Republic of Korea.
Mushroom Science Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Republic of Korea.
Bioengineered. 2025 Dec;16(1):2458376. doi: 10.1080/21655979.2025.2458376. Epub 2025 Jan 29.
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 gene in 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.
基因编辑正在成为一种在蘑菇中引入新功能的强大工具。虽然CRISPR/Cas9诱导的双链断裂（DSB）通常依靠非同源末端连接（NHEJ）来破坏基因，但在蘑菇中精确插入异源DNA的研究较少。在这里，我们评估了在Cas9-gRNA核糖核蛋白（RNP）诱导的DSB处插入有或没有同源臂的供体DNA（8-1008碱基对）的效果。将供体DNA与靶向该基因的RNP共转化到中，产生了184个没有同源臂的转化体和781个带有300碱基对同源臂的转化体（HR_供体DNA）。限制性分析和测序鉴定出122个具有完整供体DNA序列的HR_供体DNA转化体，实现了15.6%的同源定向修复（HDR）效率（122/781），相比之下，184个转化体中通过NHEJ有8个实例。这些发现突出了HDR在蘑菇中进行精确基因组编辑的可行性，能够进行靶向修饰以增强功能。
