Modulating Pathogenesis with Mobile-CRISPRi

Qu, Jiuxin; Prasad, Neha; Yu, Michelle; Chen, Shuyan; Lyden, Amy; Herrera, Nadia; Silvis, Melanie R.; Crawford, Emily; Looney, Mark R.; Peters, Joseph E.; Rosenberg, Oren S.

doi:10.1128/jb.00304-19

Cited by 33 publications

(27 citation statements)

References 56 publications

(61 reference statements)

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“…We have recently applied CRISPRi technology to systematically query the importance of approximately 13,000 genomic features of E. coli in different conditions [69]. CRISPRi technology has been extended to different organisms to study essential genes [70][71][72][73][74][75][76][77][78] and has been recently applied to E. coli to uncover host factors involved in T4, 186, and λ phage infection [52]. Lastly, Dub-seq uses shotgun cloning of randomly sheared DNA fragments of a host genome on a dual-barcoded replicable plasmid and next-generation sequencing to map the barcodes to the cloned genomic regions.…”

Section: Introductionmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape in E. coli

et al. 2020

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide lossof-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can

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Section: Introductionmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape in E. coli

et al. 2020

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“…We have recently applied CRISPRi technology to systematically query the importance of ~13,000 genomic features of E. coli in different conditions (Rishi et al 2020 submitted). CRISPRi has been applied to different organisms to study essential genes [68][69][70][71][72][73][74][75][76], and has been recently applied to E. coli to uncover host factors involved in T4, 186 and λ phage infection [51]. Lastly, Dub-Seq uses shotgun cloning of randomly sheared DNA fragments of a host genome on a dual-barcoded replicable plasmid and next-generation sequencing to map the barcodes to the cloned genomic regions.…”

Section: Introductionmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape inE. coli

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and highthroughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phagemediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.3 Introduction:

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“…The mobile CRISPRi system makes use of two different ways of bacterial conjugation: the bacterial Tn7 transposon system for Gram‐negative bacteria [87,88] and the integrative and conjugative elements in B. subtilis (ICEBs1) for Gram‐positive bacteria [87,89]. Additionally, mobile CRISPRi was applied to control the gene expression of conditionally essential genes in Pseudomonas aeruginosa , and CRISPRi of a gene encoding the type III secretion system activator ExsA blocked effector protein secretion in culture and attenuates its virulence in mice [90].…”

Section: Gaining Physiological Insights From Crispri Screensmentioning

confidence: 99%

Impact of CRISPR interference on strain development in biotechnology

Schultenkämper

Brito

Wendisch

2020

Biotech and App Biochem

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Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

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Modulating Pathogenesis with Mobile-CRISPRi

Cited by 33 publications

References 56 publications

High-throughput mapping of the phage resistance landscape in E. coli

High-throughput mapping of the phage resistance landscape in E. coli

High-throughput mapping of the phage resistance landscape inE. coli

Impact of CRISPR interference on strain development in biotechnology

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