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.
Highlights
Bacteria utilize an assortment of anti-phage defense mechanisms, including two that act at the nucleic acid level: restrictionmodification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems
Bioinformatics search We derived a list of 1749 unique spacers from several sources: 49 E. coli strains with CRISPR structures in the CRISPRdb database, 72 strains in the ECOR collection [9], 263 strains isolated from humans or animals in various regions of France [10], and 194 Shiga toxin-producing E. coli (STEC) strains [11]
Since CRISPR-Cas systems and phages of E. coli have been better studied than those of the other bacterial hosts, we focused on 1749 unique E. coli spacers in available array sequences from the ECOR collection, Shiga toxin-producing E. coli (STEC), and other databases
Summary
Bacteria utilize an assortment of anti-phage defense mechanisms, including two that act at the nucleic acid level: restrictionmodification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems. CRISPR-Cas systems function as endonucleases, though unlike restriction enzymes, their recognition sites are programmable by CRISPR RNAs (crRNAs) [3]. As an adaptive immune system, CRISPR-Cas components incorporate fragments of DNA from invading viruses or plasmids into arrays composed of spacers interspersed with repeats on the genome [4,5]. In Type II CRISPR systems, transcribed arrays are processed into crRNAs that form a complex with the RNA-guided Cas nuclease and a trans-activating RNA (tracrRNA) [6]. The crRNA guides the complex to double-stranded DNA ‘‘protospacer’’ sequences that match the sequence of the spacer and are flanked by a ‘‘protospacer adjacent motif’’ (PAM) unique to the CRISPR system [7]. If spacer-protospacer base-pairing is a close match, Cas cuts both strands of DNA, often eliminating the plasmid or phage. We sought to determine whether various DNA modifications known to block restriction systems can impede CRISPR-Cas defenses
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