Heterokaryosis, the main obstacle in the generation of PPO1-edited Agaricus bisporus by CRISPR/Cas9 system

PPO1 gene editing in Agaricus bisporus was carried out by CRISPR/Cas9 through Agrobacterium tumefaciens-mediated transformation (ATMT). The ATMT to the gill tissue yielded 52 transformants, however, sequencing of which failed to detect any distinct PPO1 modification. Instead, two transformants, N9 and N14, were identified to have mixed sequences at the vicinity of the protospacer adjacent motif (PAM), which was attributed to the presence of multiple nuclei in the mycelial cell. Heterokaryosis of N9 was disrupted to generate 24 homokaryotic and 29 heterokaryotic isolates. PPO1 sequencing of the 53 isolates identified three edited homokaryotic isolates with two single base deletions (L18 and L22) and a four-base deletion (L41) at the 4th base from PAM, and six isolates with mixed sequences. The mixed sequences were resolved to consist of the edited and unedited PPO1s, indicating the presence of both edited and unedited nuclei, thus emphasizing that heterokaryosis is a major obstacle in generating edited nuclei in A. bisporus. Notably, the unedited PPO1s in the homokaryotic L32 and heterokaryotic L49 were discovered to be edited by single base deletion after prolonged incubation, suggesting the transfer of the Cas9-gRNA complex to neighboring nuclei in the common cytoplasm. Lastly, the PPO1-edited heterokaryon Het1, created by mating the edited homokaryotic L18 with the wild-type NH1, exhibited significantly less browning than the wild-type strain, indicating a functional role for PPO1 in the button mushroom browning.