Materials and Methods

Bacteriophage T4 Escapes CRISPR Attack by Minihomology Recombination and Repair

MATERIALS AND METHODSPlasmids. CRISPR-LbCas12a/Cas9 plasmids were constructed using the streptomycin-resistant plasmid DS-SPCas as the starting plasmid (Addgene no. 48645). Sequences of spacers (listed in Table S1 in the supplemental material) were cloned into plasmid DS-SPCas in E. coli DH5α by overlap extension PCR (Thermo Fisher Phusion High-Fidelity PCR Master Mix) as previously described (9). Transformants were selected on streptomycin plates (50 μg/ml). The spacer-containing CRISPR-Cas9/Cas12a plasmids were extracted from the transformants, and the insertion of spacer sequences was confirmed by sequencing (Retrogen).TABLE S1Spacer sequences. Download Table S1, DOCX file, 0.01 MB.Copyright © 2021 Wu et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.The pET28b vector was used for construction of homologous donor plasmids to generate uvsX.del and uvsY.del mutant phages. For uvsX.del donor plasmid, two rounds of PCR were performed as previously described (9, 71). In the first round, the two homologous arms were amplified with primers listed in Table S2. In the second round, the two fragments were stitched to each other by including a 23-bp complementary region, where the uvsX deletion (Q52-G211) was introduced into, to form a full-length donor DNA. The donor DNA was then ligated into pET28b vector at the BglII and XhoI enzyme sites. A similar strategy was used to construct the uvsY.del (D5-F133) donor plasmid using primers shown in Table S2.TABLE S2Primers used for the genetic signature analysis of CRISPR escape plaques. Download Table S2, DOCX file, 0.01 MB.Copyright © 2021 Wu et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Bacteria and bacteriophages. E. coli strains B834 (hsdRB hsdMB met thi sup0 recA+) was used for propagation of wild-type (WT) T4 phage, uvsX.del, and uvsY.del mutant phages and as plating bacteria to test the plating efficiency of various spacers (efficiency of plating of WT T4 phage EOPWT T4). E. coli DH5α hsdR17(rK– mK+) sup2 recA was used for plasmid construction and spacer plating efficiency testing.The WT T4 phage was prepared from our laboratory stock. The uvsX.del and uvsY.del mutant phages were constructed by CRISPR-Cas9 strategy in WT T4 phage background as described previously (9, 72). The spacer-containing CRISPR-Cas9 plasmid and the corresponding homologous donor plasmid were cotransformed into E. coli B834. E. coli cells either transformed with the donor plasmid or with the spacer-containing CRISPR-Cas9 plasmid were used as controls. E. coli cells containing spacer and donor plasmids and control E. coli cells were infected with WT T4 phage and the first generation (G1) recombined plaques were picked in 200 μl Pi-Mg buffer (26 mM Na2HPO4, 68 mM NaCl, 22 mM KH2PO4, 1 mM MgSO4 pH 7.5). Each plaque was purified under CRISPR pressure (G2). A single plaque from the G2 plate was picked into 200 μl Pi-Mg buffer to make the zero stock, and the mutated region was amplified by PCR and sequenced.Plaque assays. The plaque assay was performed to determine the efficiency of spacer-expressing CRISPR E. coli to restrict T4 phage infection. As described previously (9, 23), serial dilutions of WT T4 phage (∼103 to 106 PFU) were added to E. coli (∼108 cells/ml). The mixture (300 μl) was incubated for 7 min at 37°C, and then 3 ml of 0.7% top agar with streptomycin (50 μg/ml) was added, and the mixture was poured onto LB plate. After incubation at 37°C overnight, the first generation (G1) plaques were counted. The EOP (efficiency of plating) refers to the value determined by dividing the number of plaques produced by WT phage infection of CRISPR E. coli by the number of plaques produced by infection of control E. coli lacking the CRISPR plasmid.Evolutionary signatures of CRISPR escape plaques. The evolutionary signature of each plaque was examined by PCR and DNA sequencing. Briefly, individual G1 CRISPR escape plaques were picked and put into 200 μl of Pi-Mg buffer, and 0.5 μl of each was used as a template for PCR amplification with a pair of primers flanking the protospacer target site (Table S2). The amplified DNAs were electrophoresed on an agarose gel, and individual bands from the agarose gel were sliced, the DNAs were extracted using QIAquick Gel Extraction kit (Qiagen) and sequenced (Retrogene). The sequences were then aligned with the WT sequence by BioEdit software to determine the mutation(s) introduced into phage genome.Minihomology sequence analyses. Minihomology sequences flanking the CRISPR-cas9 cleavage sites were detected using the computing engine FAIR (Finding All Internal Repeats) ( The input sequences were stretches of genomic DNA covering the range of the longest deleted region for each spacer. All internal repeats of 3 or more nucleotides found within the input sequence were considered potential minihomology sites. The minihomologies were then sorted out by length of the sequence, GC content, distance to the PAM site, and frequency of usage, by SnapGene and Photoshop software programs.

Article TitleBacteriophage T4 Escapes CRISPR Attack by Minihomology Recombination and Repair


Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival.

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