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: 10470172
• 负责人: Wenyan Jiang
• 金额: $ 10.49万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470172
• 关键词: Address; Aminoglycosides; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; CRISPR interference; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cells; Chemosensitization; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Complex; Computer Analysis; Data; Development; Drug Combinations; Drug resistance; Engineering; Escherichia coli; Essential Genes; Event; 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; Public Health; Quinolone Antibiotic; RNA library; Recurrence; Resistance; Site; Staphylococcus aureus; Surveys; System; Techniques; Technology; Time; Treatment Failure; Work; antibiotic tolerance; antimicrobial; bacterial genetics; base; 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 drug combination; overexpression; rational design; resistance factors; resistance mechanism; resistant strain; screening; small molecule; 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.

项目概要 抗生素耐药性是当今公众健康面临的最大威胁之一。 耐药性极其复杂，既有遗传成分，也有非遗传成分，例如，短暂的。 细菌亚群中转录重编程（非遗传）对抗生素的耐受性可能有助于 突变（遗传）的最终增加赋予耐药性，导致反复治疗失败和 临床上出现的多药耐药性已经在适应性耐药和耐药性的病例中出现。 影响抗生素的遗传和转录决定因素的系统级调查。 敏感性将为开发新型抗菌策略奠定坚实的基础。 鉴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 先前的研究利用转座子诱变来对抗耐药性。 然而，每个非必需基因对许多细菌物种的抗生素敏感性都有贡献。 不可逆的扰动和无法靶向必需基因，转座子诱变对于 研究具有短暂性、非遗传成分（例如持久性）的表型。 针对这一挑战，我建议使用新型全基因组 CRISPR 干扰开发一个系统框架 (CRISPRi) 筛选技术来探究抗生素的遗传和转录决定因素 与使用传统设计生成的低多样性向导 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 我的初步结果表明，这种方法可以大大减少费用、劳动力和时间。 生成 CRISPR 文库所需，同时大幅提高其多样性和敏感性， 揭示了以前与抗生素敏感性无关的新基因位点。 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，这将使我们能够进行更多的调查。 更广泛的健康景观，关键包括使用这一提议的温和抑制。 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，我将进行 对抗生素敏感性的遗传和转录决定因素进行系统级研究，以及 基础和临床的两种进化不同细菌中尚未研究的基因水平附带敏感性 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能够得到证实。 大大扩展我们对抗生素多种遗传和非遗传机制的了解 耐药性并产生药物开发的新靶标。