PMID:26081634

Citation	Bryson, AL, Hwang, Y, Sherrill-Mix, S, Wu, GD, Lewis, JD, Black, L, Clark, TA and Bushman, FD (2015) Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9. MBio 6:e00648
Abstract	The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack.
Links	PubMed PMC4471564 Online version:10.1128/mBio.00648-15
Keywords	Bacteriophage T4/genetics; Bacteriophage T4/growth & development; CRISPR-Cas Systems; Cytosine/chemistry; Cytosine/metabolism; DNA, Viral/chemistry; DNA, Viral/genetics; DNA, Viral/metabolism; Escherichia coli/virology; Microbial Viability; Molecular Sequence Data; Sequence Analysis, DNA; Viral Plaque Assay
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:A0A023ZW86		GO:0016021: integral component of membrane	ECO:0000314:		C		Authors align full genome with NCBI T4 genome and this protein is identified. Authors deposit new genome in Genbank.	complete CACAO 12093

References

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