Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok, 10330, Thailand.
Center of Excellence for Innovative Diagnosis of Antimicrobial Resistance, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
Sci Rep. 2024 Oct 30;14(1):26116. doi: 10.1038/s41598-024-77442-2.
CRISPR-dead Cas9 interference (CRISPRi) has become a valuable tool for precise gene regulation. In this study, CRISPRi was designed to target the inhA gene of Mycobacterium smegmatis (Msm), a gene necessary for mycolic acid synthesis. Our findings revealed that sgRNA2 induced with 100 ng/ml aTc achieved over 90% downregulation of inhA gene expression and inhibited bacterial viability by approximately 1,000-fold. Furthermore, CRISPRi enhanced the susceptibility of M. smegmatis to isoniazid and rifampicin, which are both 50% and 90% lower than those of the wild-type strain or other strains, respectively. This study highlights the ability of CRISPRi to silence the inhA gene, which impacts bacterial viability and drug susceptibility. The findings provide valuable insights into the utility of CRISPRi as an alternative tool for gene regulation. CRISPRi might be further assessed for its synergistic effect with current anti-tuberculosis drugs and its possible implications for combating mycobacterial infections, especially drug-resistant tuberculosis.
CRISPR 失活 Cas9 干扰（CRISPRi）已成为精确基因调控的一种有价值的工具。在这项研究中，CRISPRi 被设计用于靶向分枝杆菌（Msm）的 inhA 基因，该基因为合成分枝酸所必需。我们的研究结果表明，100ng/ml aTc 诱导的 sgRNA2 可使 inhA 基因表达下调超过 90%，并使细菌活力抑制约 1000 倍。此外，CRISPRi 增强了分枝杆菌对异烟肼和利福平的敏感性，与野生型菌株或其他菌株相比，分别降低了 50%和 90%。这项研究强调了 CRISPRi 沉默 inhA 基因的能力，这会影响细菌活力和药物敏感性。这些发现为 CRISPRi 作为基因调控的替代工具提供了有价值的见解。CRISPRi 的协同作用可能会进一步评估其与目前抗结核药物的协同作用，以及其对治疗分枝杆菌感染，特别是耐药性结核病的可能影响。
