Effects of Heterokaryosis on Gene Editing of Agaricus bisporus Using CRISPR/Cas9 System
Effects of Heterokaryosis on Gene Editing of Agaricus bisporus Using CRISPR/Cas9 System
Yeon-Jae Choi1, Youn-Lee Oh2 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 of Agaricus bisporus to delete a polyphenol oxidase gene (PPO1) was conducted by Agrobacterium tumefaciens-mediated transformation (ATMT). By ATMT to the gill tissue, we isolated 52 transformants with T-DNA integration. However, despite the expression of Cas9 protein and gRNA, it wasn’t possible to detect the PPO1-edited isolates. We assume that this phenomenon is due to heterokaryosis, the presence of multiple nuclei in the mycelial cells. Heterokaryosis of the transformant was disrupted by protoplasting and selection, generated 53 isolates surviving selective conditions. Sequencing of PPO1 yielded three edited homokaryotic isolates with single base deletion (L18 and L28) and four base deletion (L41), as well as six isolates (L1, L2, L22, L32, L36, and L49) with mixed sequences. By further analysis, it was confirmed that the six mixed sequences were made with edited and wild type- PPO1. Interestingly, after prolonged incubation wild-type nuclei of L32 and L49 had been edited, and it suggest the transfer of the Cas9-gRNA complex to other nuclei. Lastly, homokaryotic isolate L18 was mated with NH1, the wild-type dikaryon strain, to generate PPO1-edited heterozygotic heterokaryon Het1. Cultivation of Het1 resulted in a significant reduction in browning, indicating that PPO1 plays a role in the browning of the button mushroom. Deep sequencing targeting PPO1 in the Het1 fruiting body showed the presence of PPO1:ppo1 in 1:1 ratio. However, the integrated chromosomes were segregated unequally during sporulation potentially due to the burden of maintenance. [Supported by New Breeding Technologies Development Program Project No. RS-2022-RD010233, RDA, Korea]
