Heterokaryosis, the main obstacle in the generation of PPO1-edited Agaricus bisporus by CRISPR/Cas9 system
Heterokaryosis, the main obstacle in the generation of PPO1-edited Agaricus bisporus by CRISPR/Cas9 system
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DOI：
10.1016/j.scienta.2023.112095
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发表时间：
2023-04-25
影响因子：
4.3
通讯作者：
Ro，Hyeon-Su
中科院分区：
文献类型：
--
作者：
Choi，Yeon Jae;Eom，Hyerang;Ro，Hyeon-Su
通过农杆菌介导的转化（ATMT），利用 CRISPR/Cas9 对双孢蘑菇进行 PPO1 基因编辑。鳃组织的 ATMT 产生了 52 个转化体，但测序未能检测到任何明显的 PPO1 修饰。相反，两个转化体 N9 和 N14 被鉴定在原型间隔子相邻基序 (PAM) 附近具有混合序列，这归因于菌丝细胞中存在多个核。 N9 的异核性被破坏，产生 24 个同核分离株和 29 个异核分离株。对 53 个分离株进行 PPO1 测序，鉴定出 3 个经过编辑的同核分离株，其中两个单碱基缺失（L18 和 L22），以及 PAM 的第 4 个碱基处有一个四碱基缺失 (L41)，以及 6 个具有混合序列的分离株。混合序列被解析为由编辑和未编辑的PPO1组成，表明编辑和未编辑的细胞核都存在，从而强调异核现象是在A中生成编辑的细胞核的主要障碍。双孢菌。值得注意的是，在长时间孵育后，同核L32和异核L49中未编辑的PPO1被发现被单碱基删除所编辑，这表明Cas9-gRNA复合物转移到共同细胞质中的邻近细胞核。最后，通过将编辑的同核生物 L18 与野生型 NH1 交配而产生的 PPO1 编辑的异核体 Het1，其褐变程度明显低于野生型菌株，表明 PPO1 在蘑菇褐变中的功能性作用。
PPO1gene editing inAgaricus bisporuswas carried out by CRISPR/Cas9 throughAgrobacterium tumefaciens-mediated transformation (ATMT). The ATMT to the gill tissue yielded 52 transformants, however, sequencing of which failed to detect any distinctPPO1modification. 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.PPO1sequencing 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 uneditedPPO1s, indicating the presence of both edited and unedited nuclei, thus emphasizing that heterokaryosis is a major obstacle in generating edited nuclei inA. bisporus. Notably, the uneditedPPO1s 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, thePPO1-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 forPPO1in the button mushroom browning.