Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes

Vlot, Marnix; Houkes, Joep; Lochs, Silke J A; Swarts, Daan C.; Zheng, Peiyuan; Künne, Tim; Mohanraju, Prarthana; Anders, Carolin; Jínek, Martin; Oost, John van der; Dickman, Mark J.; Brouns, Stan J. J.

doi:10.1093/nar/gkx1264

Cited by 66 publications

(56 citation statements)

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“…Most sgRNAs used in this study showed low activity (Fig. 3a), and some DNA modifications on the genome of phages could also largely reduce the activity of sgRNA (17,33). Therefore, the fact that sgRNAs with low activity can be used for gene editing is significant and will greatly expand the editable locations of phage genome based on CRISPR technology.…”

Section: Resultsmentioning

confidence: 89%

“…We failed to find a difference in sequence characteristics with high-activity and low-activity sgRNA, indicating that there are other inhibitors. For example, some DNA modifications on phage genomes and the DNA target site accessibility in eukaryotic cells have been proven to influence the activity of CRISPR/Cas9 (18,33). Therefore, a more complete investigation of the on-target activity of sgRNA on phages may be necessary.…”

Section: Resultsmentioning

confidence: 99%

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Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Shen

Zhou

Chen

et al. 2018

J Virol

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is one of the most common nosocomial opportunistic pathogens and usually exhibits multiple-drug resistance. Phage therapy, a potential therapeutic to replace or supplement antibiotics, has attracted much attention. However, very few phages have been well characterized because of the lack of efficient genome-editing tools. Here, Cas9 from and a single guide RNA (sgRNA) were used to modify a virulent bacteriophage, phiKpS2. We first evaluated the distribution of sgRNA activity in phages and proved that it is largely inconsistent with the predicted activity from current models trained on eukaryotic cell data sets. A simple CRISPR-based phage genome-editing procedure was developed based on the discovery that homologous arms as short as 30 to 60 bp were sufficient to introduce point mutation, gene deletion, and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the abovementioned strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy. In the present study, we have addressed efficient, time-saving, and cost-effective CRISPR-based phage genome editing of phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proven to be enough to introduce point mutation, small frameshift deletion, gene deletion, and swap into phages, and weak sgRNAs were proven useful for precise phage genome editing but failed to select random recombinants, all of which makes the CRISPR-based phage genome-editing method easier to use.

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Shen

Zhou

Chen

et al. 2018

J Virol

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“…A lack of protection by CRISPR immunity is not limited to jumbophages: E. coli strains carrying Type I-E CRISPR-Cas that were engineered to carry a single targeting spacer against different phages revealed a lack of protection against phages R1-37 (a giant phage) and T4 (125). The ability of phage T4 to by-pass Type I-E CRISPR immunity is at least in part attributable to their genome containing glucosyl-5-hydroxymethylcytosine instead of cytosine (126), and this cytosine modification also confers infectivity to the phage when bacteria have Type II-A CRISPR-based immunity (126,127), but not when they have Type V-A CRISPR-based immunity (126). Type I-E CRISPR-Cas offers protection against phage T7, but only under low phage densities; at high MOIs the cultures were lysed as efficiently as uninduced controls.…”

Section: -Temperate Phagementioning

confidence: 99%

How important is CRISPR-Cas for protecting natural populations of bacteria against infections by mobile genetic elements?

Westra

Levin

2020

Preprint

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Articles on CRISPR commonly open with some variant of the phrase 'these short-palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other deleterious ("badass") DNAs'.There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro.Not at all clear are the ubiquity, magnitude and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes, and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical-computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea and the mobile genetic elements that infect them. SignificanceThere is no question about the importance and utility of CRISPR-Cas for editing and modifying genomes. On the other hand, the mechanisms responsible for the evolution and maintenance of these systems and the magnitude of their importance to the ecology and evolution of bacteria, archaea and their infectious DNAs, are not at all clear. With the aid of heuristic mathematical/simulation models and reviews of the existing literature, we raise questions that have to be answered to elucidate the contribution of selection -mediated by phage and plasmids -to the evolution and maintenance of this adaptive immune system and its consequences for the ecology and evolution of prokaryotes and their viruses and plasmids.

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“…T4 DNA ligase has a central role in the replication and repair of the phage genome during its infection of E. coli [43]. This also entails coping with DNA modifications such as the full substitution of cytosine for 5-hydroxymethylcytosine and glucosyl-5-hydroxymethylcytosine that naturally occurs in vivo [44,45]-this is a phage defence mechanism discussed below. Although its structure has only recently been determined [46], the mechanism of action of this enzyme has long been characterised [47].…”

Section: Common Molecular Biology Tools and Orthogonalitymentioning

confidence: 99%

Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

Pinheiro¹

2020

Bacteriophages - Perspectives and Future

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The long association and intense competition between bacteria and their viruses have created a fertile ground for evolution to develop numerous tools for DNA modification, assembly and degradation. Many of these tools underpin the past 50 years of molecular biology, and others show great potential in shaping the next 50 years of the field. Here, I present some of the tools that have come out of the bacteria-bacteriophage arms race and discuss some of the concepts that may shape their future use. Molecular biology remains a fast-growing area increasingly limited solely by researcher ingenuity.

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Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes

Cited by 66 publications

References 65 publications

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

How important is CRISPR-Cas for protecting natural populations of bacteria against infections by mobile genetic elements?

Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

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