Materials and Methods

Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

MATERIALS AND METHODSPropagation of phage strains. Manipulation of phage T4 was carried out as described in reference 62. Phage T4(glc-HMC), T4(HMC), and T4(C) were provided by Lindsay Black. For their genotypes, see Table S1 in the supplemental material. T4(C) contains amber mutations in several DNA-modifying genes (see Table S1). The amber mutations are known to revert easily, so T4(C) was propagated in the amber suppressor strain E. coli CR63 to prevent genotype reversion. Experiments with T4(C) were carried out in nonsuppressor E. coli strain DH10B to ensure that cytosines in T4(C) were unmodified. Experiments and propagation of T4(glc-HMC) and T4(HMC) were carried out with DH10B. Experiments with T4(IP0) were carried out with E. coli B834.CRISPR system and spacer design. Design of CRISPR spacers was carried out with custom code in R (see Text S1 in the supplemental material). CRISPR-targeting plasmids were constructed with the system described by L. Marraffini and coworkers (27), which consists of two plasmids, pCas9 and pCRISPR. pCas9 contains the Cas9 nuclease and tracrRNA (Addgene no. 42876). Spacers in this study were cloned into the CRISPR array on pCRISPR (Addgene no. 42875) with the Marraffini lab protocol available at Addgene. Comparison of our work with that of Yaung et al. was carried out with plasmids DS-SPCas (Addgene no. 48645) and PM-SP!TB (Addgene no. 48650) and plasmids provided by Yaung et al. For the oligonucleotides used for cloning, see Table S2 in the supplemental material.Plasmid transformation assays. The T4 protospacer and PAM sequences used in this study were individually cloned into pUC19 plasmids. One hundred nanograms of protospacer/PAM-containing pUC19 was transformed into chemically competent E. coli DH10B containing a CRISPR-Cas9 system targeting the corresponding protospacer. As a transformation control, 100 ng of pUC19 without a protospacer was transformed into DH10B containing a CRISPR-Cas9 expression system. Transformation mixtures were incubated at 37°C for 1 h in 200 µl of S.O.C. medium without antibiotic selection and then plated on LB plates containing carbenicillin at 100 µg/ml to select for pUC19. Efficiency of transformation was determined by dividing the number of CFU observed in the protospacer-containing pUC19 transformation mixture by the number of CFU observed in the control pUC19 transformation mixture.Plaque assays. Plaque assays were used to determine the ability of phage to infect DH10B bacteria containing the CRISPR-Cas9 system. Up to 104 PFU of phage in a volume of 10 µl were added to 200 µl of log-phase E. coli DH10B and incubated at room temperature for 10 min. Three milliliters of 0.4% LB top agarose was added to the bacterium-phage mixture, mixed, and poured onto LB plates containing the appropriate antibiotics (100 µg/ml kanamycin for pCRISPR, 50 µg/ml chloramphenicol for pCas9, 100 µg/ml ampicillin for DS-SPcas, and 50 µg/ml chloramphenicol for PM-SP!TB). Plates were incubated at 37°C overnight. Three biological replicates, each with three technical replicates, were prepared per experiment. The efficiency of plating was determined by dividing the number of plaques on an experimental plate by the number of plaques on a control plate containing E. coli with no CRISPR system. A Kruskal-Wallis nonparametric comparison of means was carried out with GraphPad Prism software for each experiment.Phage DNA isolation and sequencing. Phage lysates were grown at a multiplicity of infection of 0.01 on DH10B. Phage T4 DNAs were isolated with the Norgen phage DNA isolation kit (Norgen Biotek Corp., Thorold, Canada). Chloroform-treated phage lysates were concentrated by 4% precipitation in PEG 4000–500 mM NaCl, resuspended in Tris-EDTA buffer, and purified as recommended. The concentration of isolated T4 phage DNAs was measured with the Quant-iT PicoGreen dsDNA Assay kit (Life Technologies, Carlsbad, CA). For single-molecule sequencing, purified phage DNA was fragmented to an average size of 1.5 kb via adaptive focused acoustics (Covaris, Woburn, MA). SMRTbell template sequencing libraries were prepared as previously described (63). Sequencing was carried out on a PacBio RS II (Pacific Biosciences, Menlo Park, CA) by using P4/C2 sequencing chemistry and standard protocols for large insert libraries. Consensus sequences were generated by Quiver, and kinetic data were generated with SMRT Analysis Software v2.0 (Pacific Biosciences). For further details of the methods used, see Text S1 in the supplemental material. Libraries for the sequencing of T4(C) and T4(C)R were made with Illumina’s Nextera XT DNA Sample Preparation kit with 1 ng of input DNA, generating paired-end fragments. Metagenomic sequencing was performed on an Illumina MiSeq instrument. Paired-end reads from the MiSeq instrument were quality trimmed. Reads were aligned with the NCBI T4 genome sequence by Geneious to form consensus sequences for T4(C) and T4(C)R.Nuclease assays. One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.Nucleotide sequence accession numbers. The T4 genome sequences have been deposited in GenBank at NCBI under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"KJ477684.1","term_id":"628971780","term_text":"KJ477684.1"}}KJ477684.1 for T4(glc-HMC)/T4 wild type, {"type":"entrez-nucleotide","attrs":{"text":"KJ477685.1","term_id":"628971983","term_text":"KJ477685.1"}}KJ477685.1 for T4(HMC)/T4(147), and {"type":"entrez-nucleotide","attrs":{"text":"KJ477686.1","term_id":"628972172","term_text":"KJ477686.1"}}KJ477686.1 for T4(C)/T4(GT7).

Article TitleCovalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

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.


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