High-throughput application of CRISPR technology to identify gene function in Salmonella

高通量应用CRISPR技术鉴定沙门氏菌基因功能

• 批准号: 9172073
• 负责人: Joseph Thomas Wade
• 金额: $ 20.96万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-25 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9172073
• 关键词: Address; BCAR1 gene; Bacteria; Bacterial Genes; Bacterial Genome; Bacterial Physiology; CRISPR interference; CRISPR/Cas technology; Cessation of life; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Effectiveness; Engineering; Escherichia coli; Essential Genes; Future; Gene Deletion; Genes; Genetic Transcription; Genomic approach; Genomics; Growth; Homologous Gene; Knowledge; Methodology; Methods; Phenotype; Positioning Attribute; Salmonella; Salmonella enterica; Study models; System; Technology; Work; Yeasts; base; deletion library; gene function; gene repression; genome-wide; innovation; next generation sequencing; pathogen; promoter; tool

SUMMARY Our understanding of bacterial physiology is limited by the fact that the majority of bacterial genes are uncharacterized. Even in the best studied model bacterium, Escherichia coli K-12, >1,200 genes are completely uncharacterized, and for many of the “characterized” genes, very little functional information is known. Our ability to characterize gene functions is now outpaced by identification of new genes with unknown function. Hence, it is critical that we develop high-throughput methodologies to reliably assign gene function at a more rapid pace. Two such high-throughput methods, Chemical Genomics and SGA, have been developed for use in yeast, and subsequently applied in E. coli. However, these methods are labor-intensive, provide little information about essential genes, and are only readily applicable in species with one (for chemical genomics) or two (for SGA) deletion collections (deletion collections are available for only a few bacterial species). Furthermore, SGA is only applicable to species in which pairs of gene deletions can be easily combined. We will harness the combined power of CRISPR interference (CRISPRi) and next-generation sequencing technologies to redesign the Chemical Genomics and SGA approaches. Our preliminary data demonstrate the effectiveness of this approach in E. coli and establish CRISPRi in Salmonella. CRISPRi-based methods for Chemical Genomics and SGA have three major advantages over the established approaches. First, CRISPRi- based methods are far less labor intensive and can be applied more rapidly and more cheaply than the existing approaches. Second, CRISPRi-based methods are more effective for studying essential genes. Third, CRISPRi- based methods are readily applicable to a wide range of bacterial species, including species that lack deletion collections. We expect to identify groups of functionally related genes using each approach individually, and by combining data from both approaches. We expect that the relationships we identify between genes will be the basis of many future studies, as has been the case for Chemical Genomics and SGA data in yeast and E. coli. Moreover, our work will have a long-term impact by establishing these methods as facile, high-throughput tools for investigating gene function in a wide variety of bacterial species. The proposed work is highly significant because it provides a powerful solution to the major problem of identifying gene function. The proposed work is innovative because no prior studies have applied CRISPRi to Chemical Genomics or SGA. Moreover, there have been no previous high-throughput studies of gene function in Salmonella.

总结 我们对细菌生理学的理解受到以下事实的限制，即大多数细菌基因是 没有特征的即使在研究得最好的模式细菌大肠杆菌K-12中，也有超过1,200个基因完全被 未表征的，并且对于许多“表征的”基因，已知的功能信息非常少。我们的能力 鉴定功能未知的新基因的速度超过了对基因功能的鉴定。因此 我们开发高通量方法以更快的速度可靠地分配基因功能是至关重要的。 两种这样的高通量方法，化学基因组学和SGA，已经被开发用于酵母， 随后在E.杆菌然而，这些方法都是劳动密集型的，提供的信息很少， 必需基因，并且仅适用于具有一个（化学基因组学）或两个（SGA）的物种 删除集合（删除集合仅可用于少数细菌物种）。此外，SGA仅 适用于基因缺失对可以容易地组合的物种。 我们将利用CRISPR干扰（CRISPRi）和下一代测序的组合力量， 重新设计化学基因组学和SGA方法的技术。我们的初步数据表明， 在E.在沙门氏菌中建立CRISPRi。基于CRISPRi的 化学基因组学和SGA与现有方法相比有三大优势。首先，CRISPRi- 的方法劳动密集度低得多，可以比现有的方法更快、更便宜地应用。 接近。其次，基于CRISPRi的方法对于研究必需基因更有效。第三，CRISPRi- 基于的方法容易适用于广泛的细菌物种，包括缺乏缺失的物种 收藏. 我们希望通过单独使用每种方法以及结合使用每种方法来识别功能相关的基因组。 两种方法的数据。我们希望我们确定的基因之间的关系将是许多基因的基础。 未来的研究，酵母和大肠杆菌中的化学基因组学和SGA数据就是如此。杆菌而且我们 通过将这些方法建立为用于调查的简便、高通量工具， 基因在多种细菌物种中的功能。拟议的工作非常重要，因为它提供了 这是解决基因功能鉴定这一重大问题的有力方案。这项工作具有创新性，因为 没有先前的研究将CRISPRi应用于化学基因组学或SGA。此外，此前没有 沙门氏菌基因功能的高通量研究。