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

用于系统询问细菌抗生素敏感性的遗传和转录决定因素的新 CRISPR 工具

• 批准号: 10883888
• 负责人: Wenyan Jiang
• 金额: $ 24.9万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10883888
• 关键词: Address; Aminoglycosides; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; CRISPR interference; CRISPR library; CRISPR screen; CRISPR/Cas technology; Chemosensitization; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Complex; Computer Analysis; Cytoprotection; Data; Development; Drug Combinations; Drug resistance; Engineering; Escherichia coli; Essential Genes; Event; Expression Library; Foundations; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genomic DNA; Genomics; Growth; Guide RNA; In Vitro; Investigation; Knowledge; Libraries; Methodology; Multi-Drug Resistance; Mutagenesis; Mutate; Mutation; Oligonucleotides; Open Reading Frames; Organism; Pathway Analysis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Predisposition; Public Health; Quinolones; RNA library; Recurrence; Resistance; Site; Staphylococcus aureus; Surveys; System; Techniques; Technology; Time; Treatment Failure; Work; antibiotic tolerance; antimicrobial; bacterial genetics; beta-Lactams; cellular targeting; clinically relevant; combat; computerized tools; cost; design; drug development; experimental study; fitness; gene function; gene repression; genetic architecture; genome-wide; genomic locus; improved; in vivo; insight; loss of function; member; new therapeutic target; non-genetic; novel; novel therapeutics; overexpression; rational design; resistance factors; resistance mechanism; resistant strain; screening; small molecule; synergism; trait; transcriptional reprogramming

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.

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 耐药性极其复杂，既有遗传因素，也有非遗传因素。例如，瞬态 细菌亚群中通过转录重编程（非遗传）产生的抗生素耐受性可能有助于 最终导致耐药的突变（遗传）增加，导致复发性治疗失败， 临床出现多药耐药。这在适应性耐药性的案例中已经看到， 细菌持久性影响抗生素的遗传和转录决定簇的系统水平调查 敏感性将为开发新的抗菌策略奠定坚实的基础。特别是， 鉴定使细菌对特定抗生素敏感的因子（药物增强作用）是一种可行的策略， 面对阻力。使用转座子诱变，以前的研究已经无偏地评估了 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。但由于其 由于转座子不可逆的扰动和不能靶向必需基因，因此转座子诱变对于 研究具有暂时性、非遗传成分（如持久性）的表型。为了解决 为了应对这一挑战，我建议使用一种新的全基因组CRISPR干扰来开发一个系统框架， （CRISPRi）筛选技术来询问抗生素的遗传和转录决定簇 灵敏度与传统的基于设计的低多样性向导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的天然能力， 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，该方法可以大大减少费用、人力和时间 这是产生CRISPR文库所必需的，同时大大提高了它们的多样性和灵敏度， 从而揭示了以前与抗生素敏感性无关的新的遗传基因座。而且相比 转座子诱变引起的强烈的功能丧失扰动， 预期文库产生广泛的转录抑制。这将使我们能够调查 更广泛的健身景观，关键是包括必要基因的轻度抑制。利用这一提议， 全基因组CRISPRi文库及其诱导型版本，沿着其他技术，包括ORF 过表达文库，细菌遗传学，计算分析和动物模型，我将进行一个 对抗生素敏感性的遗传和转录决定因素进行系统水平的研究， 在基础和临床两种进化上不同细菌中研究不足的基因水平的侧支敏感性 重要性：大肠杆菌和金黄色葡萄球菌。我希望这些实验的结果 大大扩展了我们对抗生素的不同遗传和非遗传机制的认识 并为药物开发提供新的靶点。