Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi

Peters, Joseph E.; Koo, Bon Chul; Patino, Ramiro; Heussler, Gary E.; Hearne, Cameron C.; Qu, Jiuxin; Inclán, Yuki F.; Hawkins, John S.; Lu, Candy H. S.; Silvis, Melanie R.; Harden, M. Michael; Osadnik, Hendrik; Engel, Joanne N.; Dutton, Rachel J.; Grossman, Alan D.; Gross, Carol A.; Rosenberg, Oren S.

doi:10.1038/s41564-018-0327-z

Cited by 210 publications

(243 citation statements)

References 31 publications

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“…This observation is consistent with the small effect of CRISPRi targeting of mrdA (the PBP2 associated with MreBCD) on fitness in E. coli (Fig. S4), and with previous work which found that Enterobacter cloacae is also relatively unaffected by mreBCD CRISPRi targeting (36). It is unclear why E. coli and other Gram-negative bacteria are less affected by mreBCD CRISPRi targeting than B. subtilis, however transcriptional buffering through feedback may play a role.…”

supporting

confidence: 91%

Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

et al. 2019

Preprint

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Essential genes are the central hubs of cellular networks. Despite their importance, the lack of high-throughput methods for titrating their expression has limited our understanding of the fitness landscapes against which essential gene expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate specific levels of CRISPRi activity and demonstrate its broad applicability in bacteria. Using libraries of mismatched sgRNAs, we characterized the expression-fitness relationships of essential genes in Escherichia coli and Bacillus subtilis. Remarkably, these relationships co-vary by pathway and are predominantly conserved between E. coli and B. subtilis despite ~ 2 billion years of evolutionary separation, suggesting that deeply conserved tradeoffs underlie bacterial homeostasis.One Sentence Summary: Bacterial essential genes have varying responses to CRISPRi knockdown that are largely conserved across ~2 billion years of evolution.

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confidence: 91%

Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

et al. 2019

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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

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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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“…In addition, increasing the portability of bacterial CRISPRa systems can greatly improve our ability to manipulate nonmodel organisms. In combination with in situ microbiome engineering tools (Brophy et al, 2018;Ronda et al, 2019), we envision that CasTA and similar technologies (Peters et al, 2019) could modulate gene expression in a precise and programmable fashion across diverse bacterial communities to elucidate fundamental processes underlying microbial ecology and provide useful applications in the emerging field of microbiome engineering.…”

Section: Discussionmentioning

confidence: 99%

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

Fang

Cheung

et al. 2020

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Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation have been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by up to 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.

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Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi

Cited by 210 publications

References 31 publications

Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

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

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

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