Abstract：
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.
摘要：
结核分枝杆菌是结核病（TB）的主要病原体，结核病是一种古老而广泛的全球传染病，2021年有160万人丧生。抗生素耐药性（AMR）几十年来一直是一个持续的危机；2019年有495万人死亡与抗生素耐药性有关。虽然AMR是一个多方面的问题，药物发现是解决方案的紧迫部分，并且处于现代研究的最前沿。毫无疑问，高通量基因沉默技术的发展改变了结核病药物发现的前景，该技术能够对基因组中的每个基因进行询问，以及它们对健身的相对贡献，毒力,和AMR。该领域的最新进展是CRISPR干扰(CRISPRi)。该技术在抗微生物药敏试验（AST）中的应用是基础科学正在进行的研究的主题。CRISPRi技术可与高通量SPOT培养生长抑制试验（HT-SPOTi）结合使用，以快速评估和评估基因的重要性，包括非必需，有条件的必要（通过使用适当的培养条件），和必要的基因。此外,HT-SPOTi方法可以发展药物敏感性和耐药性。该技术对于合理设计基于靶标的抑制剂并希望验证其新化合物对所提出靶标的抗生素作用的主要机制的药物发现小组进一步有用。
