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 抗生素耐药性是对当今公共卫生的最大威胁之一。抗生素的基础机制 电阻非常复杂，具有遗传和非遗传成分。例如，瞬态 通过转录重编程（非基因）在细菌亚群中对抗生素的耐受性可能有助于 在突变（遗传）会议抗性的最终兴起中，导致反复治疗失败和 诊所中多药耐药性的出现。在适应性抗性和 细菌持久性。遗传和转录确定词的系统级调查会影响抗生素 敏感性将为开发新型抗菌策略带来坚实的基础。尤其， 鉴定感知细菌对特定抗生素（药物增强）的因素是可行的策略 面对阻力。使用转座子诱变，以前的研究无偏评估 每个细菌物种中每个非必需基因对抗生素敏感性的贡献。但是，由于它 不可逆的扰动和无法靶向必需基因，转座子诱变不是理想的选择 研究具有短暂的非遗传成分（例如持久性）的表型。为了解决 这个挑战，我建议使用新型的全基因组CRISPRINGERY开发系统的框架 （CRISPRI）筛选技术以询问抗生素的遗传和转录确定 灵敏度。与传统的基于设计的低多样性指南RNA（GRNA）库相比 基于阵列的寡核苷酸合成，拟议的技术利用CRISPR的自然能力 适应机制将基因组DNA转化为全面基因组CRRNA（类似于GRNA） 库。我的初步结果表明，这种方法可以大大减少费用，劳动和时间 生成CRISPR库所必需的，同时提高其多样性和敏感性 从而揭示了以前在抗生素敏感性中未实现的新型遗传基因座。而且，与 由转座子诱变引起的强大功能丧失扰动，潜水员的crrna成员 库有望创建广泛的转录表达式。这将使我们能够调查很多 更广泛的健身景观，完全包括对必需基因的轻度抑制。使用此建议 全基因组CRISPRI库和IT的诱导版本，以及包括ORF在内的其他技术 过表达的文库，细菌遗传学，计算分析和动物模型，我将执行一个 遗传和转录确定剂的系统级投资，抗生素灵敏度以及 基因和临床的两种进化不同细菌中研究不足的基因水平侧支灵敏度 重要性：大肠杆菌和金黄色葡萄球菌。我希望这些提议的实验结果 大大扩展了我们对抗生素的潜水遗传和非遗传机制的知识 抗药性和产生药物开发的新目标。