Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7

Grigonyte, Aurelija; Harrison, Charlotte; MacDonald, Paul R.; Montero-Blay, Ariadna; Tridgett, Matthew; Duncan, John N.; Sagona, Antonia P.; Constantinidou, Chrystala; Jaramillo, Alfonso; Millard, Andrew

doi:10.3390/v12020193

Cited by 27 publications

(28 citation statements)

References 58 publications

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“…Previous reports had indicated that OmpF serves as a secondary receptor to T2 (in addition to the primary receptor FadL) [127] and Cytidine monophosphate kinase (encoded by cmk) is an essential host factor in T7 phage infection [50]. Our genome-wide screens did not recapitulate these findings probably because these mutants provide an intermediate fitness in the presence of phages [128] and are swept from the population in the presence of highly resistant phage receptor mutations.…”

Section: A Crispri Screen Provides Deeper View Of Phage Resistance Dementioning

confidence: 55%

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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Section: A Crispri Screen Provides Deeper View Of Phage Resistance Dementioning

confidence: 55%

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

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…We also did not observe significant fitness scores for host factors that may bias the rate of lysogeny of the two temperate phages (186 and P2), leading to resistance of those lysogens. Our genome-wide screens did not recapitulate these findings probably because these mutants provide an intermediate fitness in the presence of phages [134] and are swept from the population in the presence of highly resistant phage receptor mutations.…”

Section: Plos Biologymentioning

confidence: 86%

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

et al. 2020

View full text Add to dashboard Cite

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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“…The editing of virulent phage genomes has remained a major challenge for phage engineering, largely due to the lack of universally applicable genetic tools or reliance on a native CRISPR-Cas system 25,26,28,[46][47][48][49][50] . While the introduction of foreign gene content into phages is relatively straightforward to perform with homologous recombination (HR), ultimately the selection or screening for these rare recombinants is limiting even in well characterized phages 48 .…”

Section: Cas13amentioning

confidence: 99%

RNA-targeting CRISPR-Cas13 Provides Broad-spectrum Phage Immunity

Adler

Hessler

Cress

et al. 2022

Preprint

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CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against invasive phages through general, non-specific RNA degradation upon complementary target transcript binding. Despite being RNA nucleases, Cas13 effectors are capable of inhibiting the infection of dsDNA phages but have only been investigated across a relatively small sampling of phage diversity. Here, we employ a systematic, phage-centric approach to determine the anti-phage capacity of Cas13 and find LbuCas13a to be a remarkably potent phage inhibitor. LbuCas13a confers robust, consistent antiviral activity regardless of gene essentiality, gene expression timing or target sequence location. Furthermore, after challenging LbuCas13a with eight diverse E. coli phages distributed across E. coli phage phylogenetic groups, we find no apparent phage-encoded limits to its potent antiviral activity. In contrast to other Class 2 CRISPR-Cas proteins, these results suggest that DNA phages are generally vulnerable to Cas13a targeting. Leveraging this effective anti-phage activity, LbuCas13a can be used seamlessly as a counter-selection agent for broad-spectrum phage editing. Using a two-step phage editing and enrichment approach, we show that LbuCas13a enables markerless genome edits in phages with exceptionally high efficiency and precision, including edits as small as a single codon. By taking advantage of the broad vulnerability of RNA during viral infection, Cas13a enables a generalizable strategy for editing the most abundant and diverse biological entities on Earth.

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Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7

Cited by 27 publications

References 58 publications

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

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

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

RNA-targeting CRISPR-Cas13 Provides Broad-spectrum Phage Immunity

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