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.

项目总结