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
- 来源:
- 关键词:AddressAminoglycosidesAnimal ModelAntibiotic ResistanceAntibioticsBacteriaCRISPR interferenceCRISPR libraryCRISPR screenCRISPR/Cas technologyChemosensitizationClinicClinicalClustered Regularly Interspaced Short Palindromic RepeatsCommunitiesComplementComplexComputer AnalysisCytoprotectionDataDevelopmentDrug CombinationsDrug resistanceEngineeringEscherichia coliEssential GenesEventExpression LibraryFoundationsGene ExpressionGene TargetingGenerationsGenesGeneticGenetic TranscriptionGenetic studyGenomeGenomic DNAGenomicsGrowthGuide RNAIn VitroInvestigationKnowledgeLibrariesMethodologyMulti-Drug ResistanceMutagenesisMutateMutationOligonucleotidesOpen Reading FramesOrganismPathway AnalysisPathway interactionsPharmaceutical PreparationsPhenotypePredispositionPublic HealthQuinolonesRNA libraryRecurrenceResistanceSiteStaphylococcus aureusSurveysSystemTechniquesTechnologyTimeTreatment FailureWorkantibiotic toleranceantimicrobialbacterial geneticsbeta-Lactamscellular targetingclinically relevantcombatcomputerized toolscostdesigndrug developmentexperimental studyfitnessgene functiongene repressiongenetic architecturegenome-widegenomic locusimprovedin vivoinsightloss of functionmembernew therapeutic targetnon-geneticnovelnovel therapeuticsoverexpressionrational designresistance factorsresistance mechanismresistant strainscreeningsmall moleculesynergismtraittranscriptional reprogramming
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,
识别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,
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。
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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