High-throughput application of CRISPR technology to identify gene function in Salmonella
High-throughput application of CRISPR technology to identify gene function in Salmonella
批准号:
9172073
负责人:
Joseph Thomas Wade
金额:
$20.96万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-06-25 至 2018-05-31
关键词:
AddressBCAR1 geneBacteriaBacterial GenesBacterial GenomeBacterial PhysiologyCRISPR interferenceCRISPR/Cas technologyCessation of lifeChemicalsClustered Regularly Interspaced Short Palindromic RepeatsDataDevelopmentEffectivenessEngineeringEscherichia coliEssential GenesFutureGene DeletionGenesGenetic TranscriptionGenomic approachGenomicsGrowthHomologous GeneKnowledgeMethodologyMethodsPhenotypePositioning AttributeSalmonellaSalmonella entericaStudy modelsSystemTechnologyWorkYeastsbasedeletion librarygene functiongene repressiongenome-wideinnovationnext generation sequencingpathogenpromotertool
中文摘要
总结
英文摘要
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.
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