Department of Respiratory Medicine, Nan'an Hospital, 330, Ximei Residential District, Xinhua Street, Quanzhou, Fujian Province, China.
The Second People's Hospital of Three Gorges University, 18, Tiyuchang Road, Yichang, Hubei Province, China.
Arch Microbiol. 2023 Dec 26;206(1):41. doi: 10.1007/s00203-023-03770-y.
Vibrio parahaemolyticus is a significant cause of seafood-associated gastroenteritis and pestilence in aquaculture worldwide. Despite extensive research, strategies for protein depletion in this pathogen remain limited. Herein, we constructed a new CRISPR interference (CRISPRi) system for gene repression based on the combination of a shuttle vector pVv3 and the nuclease-null Cas9 variant (dead Cas9, or dCas9) from Streptococcus pyrogens. This CRISPRi is induced by adding both IPTG and arabinose. We showed that gene repression is scalable via the use of multiple sgRNAs. We also demonstrated that this gene repression can be precisely tuned by adjusting the amount of two different inducers and can be reversed by removing the inducers. This system provides a simple approach for selective gene repression on a genome-wide scale in V. parahaemolyticus. Application of this system will dramatically accelerate investigations of this bacterium, including studies of physiology, pathogenesis, and drug target discovery.
副溶血弧菌是一种重要的食源性肠胃炎病原体，也是水产养殖中瘟疫的罪魁祸首。尽管已经进行了广泛的研究，但针对该病原体的蛋白质耗竭策略仍然有限。在此，我们构建了一种新的基于穿梭载体 pVv3 和无核酸酶 Cas9 变体（失活 Cas9 或 dCas9）的 CRISPR 干扰（CRISPRi）系统，用于基因抑制。该 CRISPRi 通过添加 IPTG 和阿拉伯糖诱导。我们表明，通过使用多个 sgRNA 可以实现基因抑制的扩展性。我们还证明，通过调整两种不同诱导剂的用量可以精确调节基因抑制，并且可以通过去除诱导剂来逆转。该系统为在副溶血弧菌中进行全基因组范围的选择性基因抑制提供了一种简单的方法。该系统的应用将极大地加速对该细菌的研究，包括生理学、发病机制和药物靶标发现的研究。
