MATERIALS AND METHODSPlasmids, bacteria, and bacteriophageThe spacer plasmids, 20-995, 20-1070, 23-2, and 23-1490 were constructed in a previous study (14). WT T4 phage was propagated on E. coli P301 (sup0), as previously described (38–40). T4(C) is a mutant phage containing an amber mutation at amino acid 58 of gene 42 that codes for deoxycytidine monophosphate hydroxymethylase and an amber mutation at amino acid 124 of gene 56 that codes for deoxycytidine triphosphatase (3). The T4(C) mutant was propagated on E. coli B834 (hsdRB hsdMB met thi sup0) for only one generation to prevent accumulation of spontaneous revertants. The T4(C) phage stocks containing revertant phage at a frequency of <10−6 were used in all the experiments.Plaque assaysThe efficiency of individual spacers to restrict T4 phage infection was determined by plaque assay, as previously described (14). Briefly, the CRISPR-Cas plasmids with different spacers were transformed into E. coli DH5α hsdR17(rK– mK+) sup2 individually. Up to ~107 plaque-forming units (PFU) of either WT T4 or T4(C) in 100 μl of Pi-Mg buffer (26 mM Na2HPO4, 22 mM KH2PO4, 70 mM NaCl, and 1 mM MgSO4) was added to 300 μl of E. coli (~108 cells/ml) containing the CRISPR-Cas plasmid. After a 7-min incubation at 37°C, 3 ml of 0.7% top agar with streptomycin (50 μg/ml) was added into each tube, mixed, and poured onto LB-streptomycin plates. The plates were then incubated at 37°C overnight. The EOP was calculated by dividing the PFU produced from infection of E. coli by the input PFU determined under permissive conditions.DNA sequencing of single plaquesSingle plaques were picked using a sterile Pasteur glass pipette and transferred into a 1.5-ml Eppedorf tube containing 200 μl of Pi-Mg buffer plus 2 μl of chloroform. After a 1-hour incubation at room temperature with mixing every few minutes, 4 μl of the sample was used as a template for polymerase chain reaction (PCR) using Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific). Before starting PCR, the phage was denatured at 95°C for 10 min. Amplification was performed using appropriate primers flanking the protospacer sequence. The amplified DNA was purified by agarose gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen) and sequenced (Retrogene).Selection of CEMsThree hundred microliters of E. coli DH5α containing the CRISPR-Cas plasmid (~108 cells/ml) was infected with WT T4 and mixed with 3 ml of 0.7% top agar with streptomycin (50 μg/ml) and poured onto an LB-streptomycin plate. After overnight incubation at 37°C, the plaques formed (G1) were picked by stabbing each plaque with a sterile toothpick and transferring them to another LB-streptomycin plate. The plaques formed (G2) are then subjected to the same process two to three more times (G3 to G5). Single plaques at each stage were sequenced, as described above, after amplification of the regions flanking the protospacer sequence using appropriate primers. For the 20-995 spacer, 1172-bp upstream and 226-bp downstream flanking regions were amplified; for 20-1070, 1247-bp upstream and 151-bp downstream flanking regions were amplified; for 23-1490, 227-bp upstream and 798-bp downstream flanking regions were amplified; and for 23-2, 129-bp upstream and 896-bp downstream flanking regions were amplified. E. coli DH5α without the CRISPR-Cas plasmid was used as a control.Evolution of CEMsEvolution of phages isolated from a single plaque was carried out, as shown in Fig. 3D. A single plaque was picked and transferred into a 1.5-ml Eppedoff tube containing 1 ml of Pi-Mg buffer plus 10 μl of chloroform. The phage titer was determined by plaque assay following serial dilutions. Four milliliters of log-phase E. coli DH5α cells (~2 × 108 cells/ml) containing the CRISPR-Cas plasmid was infected with phages at an MOI of 0.001 at 37°C. Three hundred fifteen minutes after infection, 400 μl of culture was collected and treated with a few drops of chloroform, and deoxyribonuclease I (7 μg/ml) and lysozyme (10 μg/ml) were then added to the sample and incubated at 37°C for 1 hour. The cell debris was removed by centrifugation of the suspension at 7000 rpm (4300g) for 10 min at 4°C. The supernatant was transferred into a new tube, and the phages were pelleted by centrifugation for 45 min at 15,000 rpm (21,130g) at 4°C. The pellet was resuspended in 200 μl of Pi-Mg buffer, serially diluted, and plated on LB plates. Ten single plaques were picked and sequenced, as described above.Coculture of spacer 20-995 CEMsAn equal number of PFU of four T4 CEMs were mixed (fig. S2) and added to 1 ml of the log-phase E. coli DH5α cells (~2 × 108 cells/ml) at an MOI of 0.001 at 37°C. Three hundred fifteen minutes after infection, 100 μl of culture was collected and treated, as described above. Ten single plaques were picked and sequenced.Plate spot testTemperature sensitivity of each phage mutant was determined by plate spot test, as previously described (3). Briefly, 300 μl of E. coli DH5α (~108 cells/ml) was mixed with 3 ml of 0.7% top agar and poured onto an LB plate. About 1 μl of phage suspension (100 to 104 PFU) was applied on the top agar plate and left for 3 to 5 min at room temperature to let the drops dry. Three identical plates were prepared and incubated overnight at 42°, 37°, and 25°C, respectively.
Bacteria and bacteriophages arm themselves with various defensive and counterdefensive mechanisms to protect their own genome and degrade the other’s. CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) is an adaptive bacterial defense mechanism that recognizes short stretches of invading phage genome and destroys it by nuclease attack. Unexpectedly, we discovered that the CRISPR-Cas system might also accelerate phage evolution. WhenEscherichia colibacteria containing CRISPR-Cas9 were infected with phage T4, its cytosine hydroxymethylated and glucosylated genome was cleaved poorly by Cas9 nuclease, but the continuing CRISPR-Cas9 pressure led to rapid evolution of mutants that accumulated even by the time a single plaque was formed. The mutation frequencies are, remarkably, approximately six orders of magnitude higher than the spontaneous mutation frequency in the absence of CRISPR pressure. Our findings lead to the hypothesis that the CRISPR-Cas might be a double-edged sword, providing survival advantages to both bacteria and phages, leading to their coevolution and abundance on Earth.