A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803
A Reversibly Induced CRISPRi System Targeting Photosystem II in the Cyanobacterium Synechocystis sp. PCC 6803
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DOI：
10.1021/acssynbio.0c00106
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发表时间：
2020-06-19
影响因子：
4.7
通讯作者：
Pakrasi, Himadri B.
中科院分区：
文献类型：
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作者：
Liu, Deng;Johnson, Virginia M.;Pakrasi, Himadri B.
蓝细菌Synechorystis sp. 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来支持其在异养条件下的生长。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，尚未在集胞藻6803中探索稳健且可逆的诱导CRISPRi系统以敲低并恢复靶基因的表达。在这项研究中，我们建立了一个严格控制的嵌合启动子，PrhaBAD-RSW，其中茶碱响应核糖开关整合到鼠李糖诱导型启动子系统。我们应用该启动子来驱动ddCpf 1（DNase-dead Cpf 1 nuclease）在CRISPRi系统中的表达，并选择PSII反应中心基因psbD（D2蛋白）作为抑制的靶基因。psbD被特异性地敲除超过95%的天然表达，导致在光合自养条件下严重抑制集胞藻6803的光系统II活性和生长。值得注意的是，诱导剂鼠李糖和茶碱的去除逆转了CRISPRi的抑制。PsbD的表达恢复后释放的镇压，加上增加的光系统II的内容和活动。集胞藻6803中这种可逆诱导的CRISPRi系统代表了研究蓝藻光合复合物生物合成的新策略。
The cyanobacterium Synechorystis 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 Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, 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 psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited photosystem II activity and growth of Synechocystis 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 Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.