Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems provide protection against viral1 and plasmid2 infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host1. These sequences, known as spacers, are transcribed into short CRISPR RNA guides3,4,5 that specify the cleavage site of Cas nucleases in the genome of the invader6,7,8. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR–Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR–Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.
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We thank R. Heler for plasmid pRH163; J. McGinn for plasmid pJM62; J. Penades for providing ϕ12 as well as assistance in working with it; B. Dujon for permission to use the I-SceI endonuclease; and The Rockefeller University Genomics Resource Center core facility for performing next-generation sequencing. J.W.M. is a Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. L.A.M. is supported by the Rita Allen Scholars Program, an Irma T. Hirschl Award, a Sinsheimer Foundation Award, a Burroughs Wellcome Fund PATH award, an NIH Director’s New Innovator Award (1DP2AI104556-01) and an HHMI-Simons Faculty Scholar Award.
The authors declare no competing financial interests.
Reviewer Information Nature thanks R. Barrangou, J. Doudna and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Figure 1 Chromosomal spacer acquisition in the S. pyogenes type II-A CRISPR–Cas locus.
a, Organization of the S. pyogenes type II CRISPR–Cas locus. Arrows indicate the annealing positions of the primers used to enrich for PCR products containing expanded CRISPR loci. R, repeat; S, new spacer. b, Abundance (RPMchr) of chromosomal sequences incorporated as spacers from wild-type cells in Fig. 1a (close-up on the dif site, the chromosomal terminus). Data previously reported for spacer acquisition by the type I CRISPR–Cas system of E. coli showed the accumulation of new spacer sequences at two hotspots adjacent to the two major ter sites at the chromosomal terminus. ter sites are not characterized in S. aureus, but the absence of multiple peaks indicates that either DSB patterns at the terminus are more distributed in S. aureus, or the nature of the breaks allows bidirectional adaptation in S. aureus but not in E. coli. c, Abundance (normalized reads) of individual spacers from the experiment in Fig. 1a, derived from the forward (top) or reverse (bottom) strand of the chromosome. Blue dots represent spacers derived from sites with a 5′-NGG-3′ PAM immediately downstream of the 3′ end of the spacer. Red dots represent spacers with a non-NGG PAM. Insert, percentage of normalized spacers with or without 3′ PAMs. d, Abundance (RPMchr) of chromosomal sequences from the experiment in Fig. 1a, derived from the forward (light blue) or reverse (olive) strand, incorporated as spacers into the CRISPR array. e, Abundance (RPMchr) of chromosomal spacers in cells harbouring wild-type cas9 following a 30-min infection with ϕ12γ3 at an MOI of 100. f, Abundance (RPMchr) of chromosomal sequences incorporated as spacers in cells with an I-sceI site with or without I-SceI expression (orange and blue, respectively; close-up of the I-sceI recognition site). g, Abundance (RPMchr) of chromosomal sequences, derived from the forward (light blue) or reverse (olive) strand, incorporated as spacers in srtA I-sceI cells from Fig. 1b (close-up of the I-sceI recognition site). sce-I, I-sceI recognition sequence; grey triangles, chi sites pointing in 5′–3′ direction, with the dotted lines marking the first chi sites upstream of the dif site.
Extended Data Figure 2 Generation of ϕ12γ3.
a, Genome organization of the staphylococcal temperate phage ϕ12. The grey arrows show the location of a bidirectional promoter controlling the expression of the lysogeny and lysis genes. The location of the cos site is also noted. ϕ12γ3 contains a deletion (red) of the cI-like repressor gene (and part of a neighbouring helicase gene) that prevents the establishment of lysogeny. b, ϕ12 forms turbid plaques owing to its ability to lysogenize and form colonies that are resistant to superinfection. By contrast, ϕ12γ3 forms clear plaques owing to its inability to lysogenize. c, Extracellular viral particles measured as p.f.u. obtained from culture supernatants 0, 15, 30, 45 and 60 min after infection with ϕ12γ3. After an initial period in which the phage particles in the medium decrease because of phage adsorption, the p.f.u. value increases after the burst of the infected cells. d, Staggered cleavage by the terminase complex at the cos site generates free DNA ends with a 10-bp 3′ overhang during DNA packaging. e, A view of ϕ12 with the cleaved cos sites at either end of the genome. During injection, the hnh (magenta) proximal end enters the bacterial cell first while the ter (orange) proximal end remains temporarily in the phage capsid.
Extended Data Figure 3 Patterns of ϕ12γ3 spacer acquisition.
a, Abundance (RPMϕ12) of ϕ12γ3 (green) or ϕ12γ3cos-flip (purple) sequences incorporated as spacers into the CRISPR array 30 min after infection at an MOI of 1. Grey triangles, chi sites pointing in 5′–3′ direction. b, Abundance (RPMϕ12) of ϕ12γ3 forward (light blue) or reverse (olive) strand sequences incorporated into the CRISPR array from the triplicate addAn experiment in Fig. 1c. c, As in b, but showing abundance in RPMϕ12 raw, which does not normalize reads to account for the GG content in each 1-kb bin. chi, first chi site upstream of the cos site. d, To corroborate the involvement of chi sites in spacer acquisition obtained after infection with ϕ12γ3 (green), we introduced two forward-facing chi sites (pink arrow and dotted line) in tandem into this phage about 2.5 kb upstream of the cos site generating ϕ12γ3chi-extra. CRISPR adaptation against this phage showed comparable levels of spacer acquisition for sequences mapping to the region between the cos site and the new chi sites. However, adaptation in the region to the left of the new chi sites, normally a highly adapted region, was reduced substantially. Abundance (RPMspc) of ϕ12γ3 (green) or ϕ12γ3chi-extra (pink) sequences incorporated as spacers into the CRISPR array 30 min after infection at an MOI of 10 is shown. e, Abundance (RPMϕ12) of ϕ12γ3 (red) or ϕ12γ3cos-flip (blue) sequences incorporated as spacers following a 30-min infection of addAn cells at an MOI of 10. Despite the absence of chi sites pointing in the 3′–5′ direction, spacer acquisition from ϕ12γ3cos-flip is limited to the area immediately adjacent to the cos site, in contrast to that observed for wild-type cells in Fig. 2g. This suggests that addAn degradation increases the number of free DNA ends used as adaptation substrates and that the cos site serves as the main entry point for the adaptation machinery.
Extended Data Figure 4 Patterns of ϕ12γ3 spacer acquisition using conventional primers or wild-type cas9.
a, Abundance (RPMϕ12) of ϕ12γ3 sequences incorporated into the CRISPR array after overnight infection at an MOI of 10. CRISPR loci within a single sample were amplified using either enrichment primers (green) or conventional primers (orange). Insert: location of the conventional primers outside the CRISPR repeats. b, Individual spacers common to both data sets in a plotted with RPMϕ12 values for enrichment primers on the y axis and conventional primers on the x axis. The diagonal dotted line indicates the identity line. c, Abundance (RPMϕ12) of ϕ12γ3 sequences incorporated into the CRISPR array after a 30-min infection at an MOI of 100 of cells harbouring hcas9 (purple) or wild-type cas9 (green). d, Individual spacers common to both data sets in c were plotted with RPMϕ12 values for hcas9 on the y axis and wild-type cas9 on the x axis. The diagonal dotted line indicates the identity line.
Extended Data Figure 5 PAM preference and strand bias for ϕ12γ3 spacer acquisition.
a, Abundance (normalized reads) of individual spacers from one of the wild-type replicates in Fig. 1c, derived from the forward (top) or reverse (bottom) strand of ϕ12γ3 following a 30-min infection at an MOI of 10. Blue dots represent spacers associated with NGG PAMs; red dots represent spacers with non-NGG flanking sequences. Insert, percentage of normalized spacers with or without canonical NGG PAMs. b, Abundance (RPMϕ12) of ϕ12γ3 forward (light blue) or reverse (olive) strand sequences incorporated into the CRISPR array from the triplicate wild type experiment in Fig. 1b. c, As in b, but showing abundance in RPMϕ12 raw, which does not normalize reads to account for the GG content in a given 1-kb bin. Grey triangles, chi sites pointing in 5′–3′ direction. d, The number of 5′-NGG-3′ PAM sites within 1-kb bins on the forward (light blue), reverse (olive) or combined (green) strands of the ϕ12γ3 genome. e, From one of the wild-type replicates in Fig. 1b, the percentage of PAMs within each 1-kb bin that are represented by at least one spacer are plotted against the ϕ12γ3 genome. This pattern of PAM acquisition demonstrates that the spacer distribution pattern of ϕ12γ3 does not result from hyper-acquisition at a few sites. Grey triangles, chi sites pointing in 5′–3′ direction.
Extended Data Figure 6 Generation and spacer acquisition profile of ϕ12 mutant phages.
a, Stages of the ϕ12 lytic cycle. Blue rhombi indicate the locations of free dsDNA ends generated in different stages that could be used for spacer acquisition. Red arrowheads indicate terminase cleavage of the cos site during viral DNA packaging. b, Mutations used in this study to halt different stages of the ϕ12 lytic cycle. c, Localization of the mutated genes in the ϕ12 genome. To eliminate polA (encoding DNA polymerase A) function we added two stop codons after the 110th codon. The function of terS (encoding the small terminase subunit) was eliminated through an in-frame deletion that left only the first and last 15 codons of the gene. The ho/ly operon was disrupted through an in-frame deletion that left the first five codons of the holin gene fused to the last seven codons of the lysin gene. d, The mutations were generated in ϕ12 prophages integrated into the S. aureus RN4220 chromosome. The resulting lysogens harbouring the mutant prophages, which were incapable of forming and releasing viral particles, were transformed with complementing plasmids carrying a wild-type copy of the mutated gene. The transformed lysogens were induced and different dilutions of the resulting lysate were spotted on plates seeded with staphylococci with or without the complementing plasmid. In all cases the mutant phages were able to lyse the complemented cells but not bacteria carrying an empty plasmid. e, Abundance (RPMϕ12) of ϕ12:polA (blue) or ϕ12:ho/ly (orange) sequences incorporated as spacers into the CRISPR array 30 min after infection at an MOI of 10. Grey triangle and dotted line mark the first 5′–3′ chi site upstream of the cos site. f, Abundance (RPMϕ12) of ϕ12:terS (red) or ϕ12:ho/ly (orange) sequences incorporated as spacers into the CRISPR array 60 min after infection at an MOI of 10. Grey triangle and dotted line mark the first 5′–3′ chi site upstream of the cos site. g, The percentage of ϕ12γ3 spacers (calculated as the ratio of ϕ12-specific spacer reads to the total spacer reads) plotted as a function of the MOI.
Extended Data Figure 7 Generation and testing of a library of spacers of ϕ12γ3 genomic DNA.
a, Engineering of the S. pyogenes type II-A CRISPR–Cas system to perform inducible spacer acquisition. The spacer acquisition genes cas1, cas2 and csn2 are under the control of a tetracycline-inducible promoter (Ptet) in one plasmid. Another plasmid contains cas9 and the tracrRNA genes along with a single-repeat CRISPR array. Spacer acquisition can be detected only via PCR (arrows represent the primers used in the reaction) in the presence of the inducer anhydro-tetracycline (aTc). b, Procedure for constructing the library. ϕ12γ3 genomic DNA was sonicated to generate fragments of about 150 bp, which were introduced through electroporation (with a water control) into cells harbouring the inducible S. pyogenes CRISPR–Cas system in the presence of aTc. This generated a library of cells containing a type II CRISPR–Cas system programmed with different spacers from the ϕ12γ3 genome. c, After transformation, cells were recovered for 3.3 h in ATC-free medium and treated with live ϕ12γ3 phage for 15 min. The surviving bacteriophage-insensitive mutant (BIMs) colonies were counted by plating 100 μl of the infected culture. Bacteria transformed with a water control (−) yielded 9 colony-forming units (c.f.u.) per 100 μl, none of which had incorporated a new spacer (data not shown). This demonstrates that at least after 3.3 h without the aTc inducer, staphylococci harbouring the engineered CRISPR–Cas system cannot acquire new spacers during subsequent infection with live phage. This rules out new events of spacer acquisition during the infection of the library with ϕ12γ3 or ϕ12γ3cos-flip. Conversely, cells transformed with sonicated ϕ12γ3 DNA yielded 327 c.f.u. per 100 μl, and all of those tested (n = 4) carried an expanded CRISPR array, indicating that substantial adaptation occurred during the electroporation of ϕ12γ3 fragments. d, Analysis of spacer selection from the library after infection. The library of spacers was treated with ϕ12γ3 or ϕ12γ3cos-flip (not shown) at MOIs of 0, 10 or 100 for 24 h to determine whether selection during phage interference could influence the spacer distribution within the library. Spacer acquisition in the uninfected library (MOI = 0) could not be detected by PCR, indicating that the majority of cells in the library did not enlarge the CRISPR array with viral spacers during this period. By contrast, strong PCR products corresponding to expanded CRISPR loci were observed following overnight phage infections, demonstrating the enrichment of adapted cells during CRISPR–Cas targeting, at MOIs of both 10 and 100. Incorporation of new spacers could not be detected by PCR in the control cells transformed with water, providing further evidence that in this assay, cells are only able to adapt during electroporation in the presence of aTc, but not after this treatment, in the absence of the inducer. S1 and S0 corresponds to CRISPR loci with and without a new spacer sequence, respectively. e, The data from Fig. 3a with the abundance (RPMϕ12 raw) of spacers derived from forward (light blue) and reverse (olive) strands of ϕ12γ3. f, As in e, showing relative (to PAM content) abundance in RPMϕ12. g, Same as Fig. 3a, showing the (RPMϕ12) values for the full phage genome. h, Same as Fig. 3b, showing the ϕ12γ3/ϕ12γ3cos-flip enrichment ratio values for the full phage genome.
Extended Data Figure 8 Design and test of spacers targeting DNA sequences adjacent to the cos site.
a, The cos site-proximal region of ϕ12γ3 or ϕ12γ3cos-flip was targeted by type II CRISPR–Cas systems programmed with a spacer matching the upstream (1, 2, 3) or downstream (4, 5, 6) region. b–g, Cells harbouring each of these CRISPR–Cas systems were infected at an MOI of 10 with either ϕ12γ3 (green) or ϕ12γ3cos-flip (purple). CRISPR–mediated survival of the cultures was monitored by measuring their optical density at 600 nm (OD600) over time.
Extended Data Figure 9 Coordination between the immunization and targeting phases of the type II CRISPR–Cas immune response.
Immunization occurs shortly after the beginning of the infection through the acquisition of new viral spacer sequences preferentially from the first free DNA end injected (green circle). Degradation of this DNA end by the AddAB nuclease, limited by chi sites, generates additional free DNA end substrates for recognition by the Cas1–Cas2–Cas9–Csn2 spacer acquisition complex and subsequent integration into the CRISPR array by the Cas1–Cas2 integrase. During targeting, Cas9 nucleases loaded with the crRNA guides generated by the acquired spacers allow the majority of the cells of the immunized host population to target the first region to be injected (green; red, last injected region) by subsequent invading viruses, providing faster and more efficient immunity.
Extended Data Figure 10 Pattern of spacer acquisition during ϕNM4γ4 infection.
a, Packaging of cos phages. In phage lambda, and presumably in ϕ12, the packaging of the viral genome invariably starts by cleavage of the terminase complex (red arrowhead) at the cos site of the phage concatamer generated by rolling circle replication, located between hypothetical genes a and z. The DNA to the left of the cos site (z gene, green circle) is the last to be packaged into the phage capsid and therefore always the first to be injected into a newly infected bacterial cell. The expected pattern of spacer acquisition starts from this dsDNA end and progressively decreases until the first chi site (yellow gradient box). b, Pattern of spacer acquisition for ϕ12γ3 10 min after infection at an MOI of 10. This is similar to the results obtained at 30 min (Fig. 1c) but comparable to the infection conditions of d. The area highlighted with a yellow gradient shows the expected spacer acquisition pattern. Grey triangles, chi sites pointing in the 5′–3′ direction, with the dotted line marking the first chi site upstream of the cos site. c, Packaging of pac phages. These phages employ a ‘headful’ DNA packaging mechanism in which each genomic concatamer is cleaved first at the pac site with subsequent cleavages occurring progressively but imprecisely, after packaging about 105% genome lengths. The exact percentage is determined by how much DNA can be filled into the phage capsid and it is always greater than 100% to ensure duplicated sequences at each end of the injected genome for recombination and circularization after infection of the next host. S. aureus pac phages and pathogenicity islands (SaPIs) display a rightward packaging mechanism, where the duplicated DNA is located downstream of the next pac site. Therefore, the last sequence to be packaged into the phage capsid and the first to be injected into a newly infected bacterial cell is variable for each infection (a, b, c gene, green circles), but lies immediately downstream of the pac site. The expected pattern of spacer acquisition starts from every different dsDNA end and progressively decreases leftward until the first chi site (yellow gradient box). d, We determined the spacer acquisition pattern of the pac phage ϕNM4γ4 (a lytic derivative of ϕNM4) 10 min after infection at an MOI of 10. As expected for the injection of variable dsDNA ends downstream of the pac site, we detected a spacer acquisition hotspot in the 10–20-kb region to the right of this site (the expected pattern is highlighted in yellow). This is consistent with the rightward migration of pac phage injection points, with 10–20 kb corresponding to the packaging of about 5–10 viral genomes, well within the observed ranges of pac phage processivity. Grey triangles, chi sites pointing in the 5′–3′ direction, with the dotted line marking the first chi site upstream of the cos site.
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Modell, J., Jiang, W. & Marraffini, L. CRISPR–Cas systems exploit viral DNA injection to establish and maintain adaptive immunity. Nature 544, 101–104 (2017). https://doi.org/10.1038/nature21719
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