CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4
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Yaung, Stephanie J., Kevin M. Esvelt, and George M. Church. 2014. “CRISPR/Cas9-Mediated Phage Resistance Is Not Impeded by the DNA Modifications of Phage T4.” PLoS ONE 9 (6): e98811. doi:10.1371/journal.pone.0098811. http://dx.doi.org/10.1371/journal.pone.0098811.Abstract
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.Other Sources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041780/pdf/Terms of Use
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