Department of Biology, Washington University, St. Louis, Missouri 63130, United States.
ACS Synth Biol. 2020 Jun 19;9(6):1441-1449. doi: 10.1021/acssynbio.0c00106. Epub 2020 May 21.
The cyanobacterium sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, P, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene (D2 protein) to target for repression. was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased photosystem II content and activity. This reversibly induced CRISPRi system in 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.
集胞藻 PCC 6803 被用作研究光合作用的模式生物，因为它可以利用葡萄糖作为唯一的碳源，在异养条件下支持其生长。CRISPR 干扰（CRISPRi）已被广泛应用于以靶向方式抑制蓝藻中基因的转录。然而，在 6803 中尚未探索出一种稳健且可逆转的诱导 CRISPRi 系统来敲低和恢复靶向基因的表达。在本研究中，我们构建了一个紧密控制的嵌合启动子 P，其中整合了茶碱响应的核糖开关到鼠李糖诱导的启动子系统中。我们应用这个启动子在 CRISPRi 系统中驱动 ddCpf1（无 DNA 酶活性的 Cpf1 核酸酶）的表达，并选择 PSII 反应中心基因（D2 蛋白）作为靶基因进行抑制。D2 蛋白的表达被特异性敲低了 95%以上，导致 6803 在光自养条件下的光系统 II 活性和生长受到严重抑制。重要的是，去除诱导剂鼠李糖和茶碱可以逆转 CRISPRi 的抑制作用。抑制解除后，PsbD 的表达恢复，伴随着光系统 II 含量和活性的增加。这种在 6803 中可诱导的 CRISPRi 系统代表了一种研究蓝藻光合作用复合物生物发生的新策略。
