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

基本信息

  • 批准号:
    10883888
  • 负责人:
  • 金额:
    $ 24.9万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

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Wenyan Jiang其他文献

Wenyan Jiang的其他文献

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{{ truncateString('Wenyan Jiang', 18)}}的其他基金

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
  • 财政年份:
    2021
  • 资助金额:
    $ 24.9万
  • 项目类别:
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:
    10215771
  • 财政年份:
    2021
  • 资助金额:
    $ 24.9万
  • 项目类别:

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