Mycobacteria Research Laboratory, Institute of Structural and Molecular Biology, School of Natural Sciences, Birkbeck, University of London, London, UK.
Division of Biosciences, College of Health, Medicine and Life Sciences, Brunel University London, Uxbridge, UK.
Methods Mol Biol. 2024;2833:23-33. doi: 10.1007/978-1-0716-3981-8_3.
Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.
结核分枝杆菌是结核病（TB）的主要病原体，这是一种古老但广泛传播的全球传染病，2021 年有 160 万人因此丧生。抗生素耐药性（AMR）几十年来一直是一个持续存在的危机；2019 年，与抗生素耐药性相关的死亡人数达到 495 万。虽然 AMR 是一个多方面的问题，但药物发现是解决方案的紧急部分，也是现代研究的前沿。
结核病药物发现的格局无疑因高通量基因沉默技术的发展而发生了转变，这些技术使人们能够检测基因组中的每个基因，以及它们对适应性、毒力和 AMR 的相对贡献。这一领域的一个最新进展是 CRISPR 干扰（CRISPRi）。该技术在抗菌药物敏感性测试（AST）中的应用是基础科学领域正在进行的研究的主题。
CRISPRi 技术可以与高通量斑点培养生长抑制测定（HT-SPOTi）结合使用，快速评估和评估基因的必需性，包括非必需、条件必需（通过使用适当的培养条件）和必需基因。此外，HT-SPOTi 方法可以开发药物敏感性和耐药性谱。
这项技术对于那些合理设计基于靶标的抑制剂的药物发现小组也很有用，他们希望验证其新型化合物针对拟议靶标对抗生素作用的主要机制。
