New CRISPR tools for systematic interrogation of genetic and transcriptional determinants of antibiotic sensitivity in bacteria

项目摘要 抗生素耐药性是当今公共卫生面临的最大威胁之一。抗生素的作用机制 抗性是极其复杂的，既有遗传成分，也有非遗传成分。例如，瞬变 通过转录重编程(非遗传)在细菌亚群中耐受抗生素可能有助于 最终导致耐药的突变(基因)增加，导致反复治疗失败和 临床出现多药耐药现象。这在适应性抵抗和 细菌的持久性。影响抗生素的遗传和转录决定因素的系统水平调查 敏感性将为开发新的抗菌策略奠定坚实的基础。特别是， 确定使细菌对特定抗生素敏感的因素(药物增强)是一种可行的策略 直面阻力。利用转座子突变，以前的研究已经不偏不倚地评估了 在许多细菌物种中，每个非必需基因对抗生素敏感性的贡献。然而，由于其 不可逆的干扰和无法靶向必需基因，转座子突变不是理想的 研究具有暂时性、非遗传性成分的表型，如持久性。为了解决这个问题 在这一挑战中，我建议使用一种新的全基因组CRISPR干扰来开发一个系统的框架 (CRISPRi)筛选技术询问抗生素的遗传和转录决定因素 敏感度。与传统的基于设计、低多样性的指南-RNA(GRNA)文库相比 基于阵列的寡核苷酸合成，所提出的技术利用了CRISPR的自然能力 将基因组DNA转化为全面的全基因组crRNA(类似于gRNA)的适应机制 图书馆。初步结果表明，这种方法可以大大减少费用、人力和时间。 产生CRISPR文库所需的资源，同时大大增加其多样性和敏感性； 从而揭示了以前没有涉及抗生素敏感性的新的遗传位点。此外，与 转座子突变引起的强烈功能丧失扰动，不同的crRNA成员 文库有望产生广泛的转录抑制。这将使我们能够调查更多 更广泛的健康前景，关键是包括对必要基因的温和抑制。使用建议的 全基因组CRISPRi文库及其诱导版本，以及包括ORF在内的其他技术 过表达文库、细菌遗传学、计算分析和动物模型，我将开展一个 对导致抗生素敏感性的遗传和转录决定因素进行系统水平的调查，以及 基础和临床两种进化不同细菌的基因水平侧支敏感性研究不足 要点：大肠杆菌和金黄色葡萄球菌。我预计这些拟议的实验结果将 大大扩展我们对抗生素的不同遗传和非遗传机制的知识 抗药性和产生药物开发的新靶点。

Project Summary Antibiotic resistance is one of the biggest threats to today’s public health. Mechanisms underlying antibiotic resistance are extremely complex and have both genetic and non-genetic components. For instance, transient tolerance of antibiotics by transcriptional reprogramming (non-genetic) in subpopulations of bacteria could aid in the ultimate rise of mutations (genetic) conferring resistance, leading to recurrent treatment failure and the emergence of multidrug resistance in the clinic. This has been seen in cases of adaptive resistance and bacterial persistence. A systems-level survey of genetic and transcriptional determinants influencing antibiotic sensitivity will generate a strong foundation for developing novel antimicrobial strategies. In particular, identification of factors that sensitize bacteria to specific antibiotics (drug potentiation) is a viable strategy to confront resistance. Using transposon mutagenesis, previous studies have unbiasedly assessed the contribution of every non-essential gene to antibiotic sensitivity in many bacterial species. However, due to its irreversible perturbation and inability to target essential genes, transposon mutagenesis is not ideal for studying phenotypes that have a transient, non-genetic component such as persistence. In order to address this challenge, I propose to develop a systematic framework using a novel genome-wide CRISPR-interference (CRISPRi) screening technology to interrogate the genetic and transcriptional determinants of antibiotic sensitivity. Compared to conventional design-based, low-diversity guide-RNA (gRNA) libraries generated using array-based oligonucleotide synthesis, the proposed technology harnesses the natural capacity of the CRISPR adaptation machinery to convert genomic DNA into comprehensive genome-wide crRNA (analogous to gRNA) libraries. My preliminary results show that this approach can greatly reduce the expense, labor and time required for the generation of CRISPR libraries, while substantially increasing their diversity and sensitivity, thereby revealing novel genetic loci not previously implicated in antibiotic sensitivity. Moreover, compared to the strong loss-of-function perturbation caused by transposon mutagenesis, the diverse crRNA members of the library are expected to create a wide range of transcriptional repression. This will allow us to survey a much broader fitness landscape, crucially including the mild suppression of essential genes. Using this proposed genome-wide CRISPRi library and an inducible version of it, along with other techniques including ORF overexpression libraries, bacterial genetics, computational analysis and animal models, I will carry out a systems-level investigation of the genetic and transcriptional determinants underlying antibiotic sensitivity, and the under-studied gene-level collateral sensitivity in two evolutionary distinct bacteria of basic and clinical importance: Escherichia coli and Staphylococcus aureus. I expect results from these proposed experiments to substantially expand our knowledge on the diverse genetic and non-genetic mechanisms of antibiotic resistance and yield novel targets for drug development.