CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4

Yaung, Stephanie J.; Esvelt, Kevin M.; Church, George M.

doi:10.1371/journal.pone.0098811

CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4

Journal Article · Mon Jun 02 00:00:00 EDT 2014 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0098811· OSTI ID:1904696

Yaung, Stephanie J. ^[1]; Esvelt, Kevin M. ^[2]; Church, George M. ^[2]

Harvard Medical School, Boston, MA (United States); Massachusetts Institute of Technology (MIT), Cambridge, MA (United States); Harvard Medical School, Boston, MA (United States)
Harvard Medical School, Boston, MA (United States)

Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N⁶-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

View Accepted Manuscript (DOE)

Research Organization:: Harvard Medical School, Boston, MA (United States); Harvard University, Cambridge, MA (United States)

Sponsoring Organization:: National Science Foundation; USDOE Office of Science (SC); Wyss Institute

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 1904696

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 6 Vol. 9; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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bacteriophages
cytosine
nucleotide sequencing
nucleotides
restriction modification systems

CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4

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