Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.
Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum.
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利用 CRISPR 干扰有效快速地灭活具核梭杆菌基因。
DOI：
10.1101/2023.09.19.558491
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Wu,Chenggang
中科院分区：
文献类型：
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作者：
Zhou,Peng;GC,Bibek;Stolte,Flynn;Wu,Chenggang
通过在具核梭杆菌中产生框内缺失突变来使基因失活是耗时的，并且大多数梭杆菌菌株在遗传上是难处理的。为了解决这些问题，我们引入了基于核糖开关的诱导型CRISPRi系统。该系统采用核酸酶失活的化脓性链球菌Cas9蛋白（dCas 9），通过恒定表达的单向导RNA（sgRNA）特异性引导至感兴趣的基因。从机制上讲，这种dCas 9-sgRNA复合物作为RNA聚合酶不可逾越的障碍，从而抑制靶基因的转录。利用这个系统，我们首先研究了两个非必需基因，ftsX和radD，关键的梭菌胞质分裂和共聚集。在加入诱导剂茶碱后，ftsX抑制引起类似于染色体ftsX缺失的丝状细胞形成，而靶向radD显著降低了RadD蛋白水平，消除了共聚集。然后将该系统扩展到探测对外膜生物发生和细胞分裂至关重要的必需基因bamA和ftsZ。令人印象深刻的是，bamA抑制破坏了膜完整性和细菌分离，阻碍了生长，而ftsZ靶向在肉汤中产生了具有受损琼脂生长的细长细胞。对F. nucleatum临床菌株CTI-2和牙周梭杆菌显示当靶向tnaA时吲哚合成减少。此外，在F.牙周炎降低ClpB，增加热敏感性。总之，我们的CRISPRi系统简化了各种梭菌菌株的基因失活。
Gene inactivation via creating in-frame deletion mutations in Fusobacterium nucleatum is time-consuming, and most fusobacterial strains are genetically intractable. Addressing these problems, we introduced a riboswitch-based inducible CRISPRi system. This system employs the nuclease-inactive Streptococcus pyogenes Cas9 protein (dCas9), specifically guided to the gene of interest by a constantly expressed single guide RNA (sgRNA). Mechanistically, this dCas9-sgRNA complex serves as an insurmountable roadblock for RNA polymerase, thus repressing the target gene transcription. Leveraging this system, we first examined two non-essential genes, ftsX, and radD, pivotal for fusobacterial cytokinesis and coaggregation. Upon adding the inducer, theophylline, ftsX suppression caused filamentous cell formation akin to chromosomal ftsX deletion, while targeting radD significantly reduced RadD protein levels, abolishing coaggregation. The system was then extended to probe essential genes bamA and ftsZ, vital for outer membrane biogenesis and cell division. Impressively, bamA suppression disrupted membrane integrity and bacterial separation, stalling growth, while ftsZ-targeting yielded elongated cells in broth with compromised agar growth. Further studies on F. nucleatum clinical strain CTI-2 and Fusobacterium periodonticum revealed reduced indole synthesis when targeting tnaA. Moreover, silencing clpB in F. periodonticum decreased ClpB, increasing thermal sensitivity. In summary, our CRISPRi system streamlines gene inactivation across various fusobacterial strains.