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

摘要 我们对细菌生理学的理解受到这样一个事实的限制，即大多数细菌基因是 没有特征的。即使在研究最好的模式细菌中，大肠杆菌K-12，&gt；1,200个基因也是完全 对于许多尚未确定特征的基因来说，人们对其功能信息知之甚少。我们的能力 现在，鉴定具有未知功能的新基因已经远远超过了对基因功能的研究。因此，它 关键是我们开发高通量的方法，以更快的速度可靠地分配基因功能。 已经开发了两种这样的高通量方法，化学基因组学和SGA，用于酵母，以及 随后在大肠杆菌中应用。然而，这些方法是劳动密集型的，提供的信息很少 基本基因，并且只适用于具有一个(化学基因组学)或两个(SGA)的物种 删除集合(删除集合仅适用于少数细菌物种)。此外，SGA只是 适用于基因缺失对容易组合的物种。 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 这种方法在大肠杆菌中的有效性，并在沙门氏菌中建立CRISPRi。基于CRISPRi的方法 与已有的方法相比，化学基因组学和SGA有三大优势。首先，CRISPRi- 与现有的方法相比，基于方法的方法的劳动强度要小得多，并且可以更快、更便宜地应用 接近了。其次，基于CRISPRi的方法在研究必需基因方面更有效。第三，CRISPRi- 基于方法的方法很容易适用于广泛的细菌物种，包括缺乏缺失的物种 收藏。 我们希望分别使用每种方法识别一组功能相关的基因，并通过组合 来自这两种方法的数据。我们预计，我们确定的基因之间的关系将成为许多 未来的研究，就像在酵母和大肠杆菌中的化学基因组学和SGA数据一样。而且，我们的 通过将这些方法建立为方便、高通量的调查工具，这项工作将产生长期影响 基因在各种各样的细菌物种中起作用。拟议的工作具有重要意义，因为它提供了 这是识别基因功能这一主要问题的有力解决方案。拟议的工作具有创新性，因为 此前没有研究将CRISPRi应用于化学基因组学或SGA。此外，以前没有过 沙门氏菌基因功能的高通量研究。

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.