Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10

Bari, S. M. Nayeemul; Walker, Forrest C.; Cater, Katie; Aslan, Barbaros; Hatoum-Aslan, Asma

doi:10.1021/acssynbio.7b00240

Cited by 63 publications

(64 citation statements)

References 58 publications

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“…Filtered lysates were mixed with LM1680-p SERP2475 overnight culture (1:1) and the mixture was plated on TSA containing 5 mM CaCl 2 using the double-agar overlay method 12 For all phage hybrids, individual plaques were isolated and re-plated three times on LM1680/p SERP-2475 to purify. Phages were propagated and their DNA was extracted as previously described 30 . Phage genomes were PCR amplified across the entire coding region for Hybrids 1-8 or gp03-gp04 for Hybrids 9-18, and the PCR products were sequenced by the Sanger method (at Eurofins MWG Operon) using the primers listed in Table S1.…”

Section: Methodsmentioning

confidence: 99%

A unique mode of nucleic acid immunity performed by a single multifunctional enzyme

Chou-Zheng

Cater

et al. 2019

Preprint

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Organisms spanning all domains of life protect against pathogens using diverse 7 mechanisms of nucleic acid immunity which detect and eliminate foreign genetic 8 material 1 . The perpetual arms race between bacteria and their viruses (phages) has given 9 rise to both innate and adaptive nucleic acid immunity mechanisms, including 10 restriction-modification and CRISPR-Cas, respectively 2 . These sophisticated systems 11 encode multiple components that sense and degrade phage-derived genetic material 12 while leaving the host genome unharmed. Here, we describe a unique mode of innate 13 immunity performed by a single protein, SERP2475, herein renamed to Nhi. We show that 14 this enzyme protects against phages by preventing phage DNA accumulation, and in a 15 purified system it degrades both DNA and RNA substrates. This enzyme also exhibits 16 ATP-dependent helicase activity, and excess ATP abrogates nuclease function, 17 suggesting a possible mechanism for its regulation. Further, using directed evolution, we 18 isolated and characterized a collection of resistant phage mutants and found that a 19 single-stranded DNA binding protein provides a natural means for phages to escape 20 immunity. These observations support a model in which Nhi senses and degrades 21 phage-specific replication intermediates. We also found that this dual-function enzyme 22 protects against diverse phages, and its homologs are distributed across several 23 bacterial phyla. Altogether, our findings reveal a new innate immune system with minimal 24 composition that provides robust defense against diverse bacterial viruses. a process that typically leads to the death of the host (Fig. 1a). In response, bacteria have 30 evolved diverse immune systems that target nearly every step of the phage infection cycle 2 --31 such systems may block phage genome entry, digest phage-derived nucleic acids, and/or 32 initiate programmed cell death, a process known as abortive infection (Abi), to prevent the 33 spread of a phage infection. Perhaps the most direct mechanism to block phage replication is 34 through the use of nucleases that sense and destroy foreign genetic material. However, bacteria 35 are known to possess only two modes of nucleic acid immunity which contribute to anti-phage 36 defense: Innate immunity provided by restriction-modification (R-M) and adaptive immunity 37 conferred by CRISPR-Cas ( Fig. 1a). Given that bacteria and their phages have been co-38 evolving for billions of years, it is only natural to expect that many unknown immune systems 39 are yet to be discovered, particularly in non-model organisms 4,5 . 40Here, we sought to identify and characterize new anti-phage immune mechanisms in the 41 commensal opportunistic pathogen Staphylococcus epidermidis RP62a 6 . This organism encodes 42 a Type III-A CRISPR-Cas system 7 , an Abi mechanism 8 , and a putative Type I R-M system 43 (GenBank Accession CP000029.1). All three systems reside within ~30,000 nucleotides of each 44 other (Extended Data 1a), in agreement with recent evidence...

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Section: Methodsmentioning

confidence: 99%

A unique mode of nucleic acid immunity performed by a single multifunctional enzyme

Chou-Zheng

Cater

et al. 2019

Preprint

Self Cite

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“…[209][210][211] In addition, by programming Cascade (together with Cas3) or Csm/Cmr complexes with self-targeting spacers in native hosts, researchers have achieved genome editing of bacterial, archaeal, and even phage genomes. [212][213][214][215] Whether similar strategies can be ported into eukaryotic cells remains to be determined, although one effort currently underway aims to treat antibiotic-resistant bacterial infections in human hosts by harnessing the programmable cell-killing activity of Cascade/Cas3. 216…”

Section: Exploring and Exploiting Crispr-cas Systemsmentioning

confidence: 99%

Harnessing “A Billion Years of Experimentation”: The Ongoing Exploration and Exploitation of CRISPR–Cas Immune Systems

Klompe

Sternberg

2018

The CRISPR Journal

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The famed physicist-turned-biologist, Max Delbrü ck, once remarked that, for physicists, ''the field of bacterial viruses is a fine playground for serious children who ask ambitious questions.'' Early discoveries in that playground helped establish molecular genetics, and half a century later, biologists delving into the same field have ushered in the era of precision genome engineering. The focus has of course shifted-from bacterial viruses and their mechanisms of infection to the bacterial hosts and their mechanisms of immunity-but it is the very same evolutionary arms race that continues to awe and inspire researchers worldwide. In this review, we explore the remarkable diversity of CRISPR-Cas adaptive immune systems, describe the molecular components that mediate nucleic acid targeting, and outline the use of these RNA-guided machines for biotechnology applications. CRISPR-Cas research has yielded far more than just Cas9-based genome-editing tools, and the wide-reaching, innovative impacts of this fascinating biological playground are sure to be felt for years to come.

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“…A number of phages have been modified with type II CRISPR systems; including phage 2972 infecting Streptococcus thermophilus, phage P2 infecting Lactococcus lactis, phiKpS2 infecting Klebsiella pneumoniae and phages T2, T4, T7 and KF1 that infect E. coli respectively [25,[27][28][29][30][31]. A type III CRISPR system has also been used to engineer phages that infect Staphylococcus aureus and Staphylococcus epidermidis [32]. Whilst CRISPR is being used for editing phage genomes, there is still much to be learned concerning what the most efficient system to use is.…”

Section: Introductionmentioning

confidence: 99%

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

Grigonyte

Harrison

MacDonald

et al. 2020

Viruses

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With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

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Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10

Cited by 63 publications

References 58 publications

A unique mode of nucleic acid immunity performed by a single multifunctional enzyme

A unique mode of nucleic acid immunity performed by a single multifunctional enzyme

Harnessing “A Billion Years of Experimentation”: The Ongoing Exploration and Exploitation of CRISPR–Cas Immune Systems

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

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