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CRISPR adaptation biases explain preference for acquisition of foreign DNA

Abstract

CRISPR–Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA (‘spacers’) are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

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Figure 1: Chromosome-scale hotspots for spacer acquisition.
Figure 2: Dependence of spacer acquisition on replication.
Figure 3: Chi sites define boundaries of protospacer hotspots.
Figure 4: Involvement of the dsDNA break repair machinery in defining spacer acquisition patterns.
Figure 5: A model explaining the preference for foreign DNA in spacer acquisition.

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Accession codes

Primary accessions

Sequence Read Archive

Data deposits

RNA sequencing data are available in the National Center for Biotechnology Information Sequence Read Archive database under accession numbers SRX862155SRX862158 in study SRP053013. Raw data of spacer sequences are accessible at http://www.weizmann.ac.il/molgen/Sorek/files/CRISPR_adaptation_2015/crispr_adaptation_2015_data.html.

References

  1. Terns, M. P. & Terns, R. M. CRISPR-based adaptive immune systems. Curr. Opin. Microbiol. 14, 321–327 (2011)

    Article  CAS  Google Scholar 

  2. Westra, E. R. et al. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 (2012)

    Article  CAS  Google Scholar 

  3. Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012)

    Article  ADS  CAS  Google Scholar 

  4. Koonin, E. V. & Makarova, K. S. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol. 10, 679–686 (2013)

    Article  CAS  Google Scholar 

  5. Sorek, R., Lawrence, C. M. & Wiedenheft, B. CRISPR-mediated adaptive immune systems in Bacteria and Archaea. Annu. Rev. Biochem. 82, 237–266 (2013)

    Article  CAS  Google Scholar 

  6. Barrangou, R. & Marraffini, L. A. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol. Cell 54, 234–244 (2014)

    Article  CAS  Google Scholar 

  7. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012)

    Article  CAS  Google Scholar 

  8. Nunez, J. K. et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nature Struct. Mol. Biol. 21, 528–534 (2014)

    Article  CAS  Google Scholar 

  9. Swarts, D. C., Mosterd, C., van Passel, M. W. & Brouns, S. J. CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7, e35888 (2012)

    Article  ADS  CAS  Google Scholar 

  10. Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat. Commun. 3, 945 (2012)

    Article  ADS  Google Scholar 

  11. Diez-Villasenor, C., Guzman, N. M., Almendros, C., Garcia-Martinez, J. & Mojica, F. J. CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli. RNA Biol. 10, 792–802 (2013)

    Article  CAS  Google Scholar 

  12. Yosef, I. et al. DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array. Proc. Natl Acad. Sci. USA 110, 14396–14401 (2013)

    Article  ADS  CAS  Google Scholar 

  13. Arslan, Z., Hermanns, V., Wurm, R., Wagner, R. & Pul, U. Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system. Nucleic Acids Res. 42, 7884–7893 (2014)

    Article  CAS  Google Scholar 

  14. Savitskaya, E., Semenova, E., Dedkov, V., Metlitskaya, A. & Severinov, K. High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli. RNA Biol. 10, 716–725 (2013)

    Article  CAS  Google Scholar 

  15. Fineran, P. C. et al. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc. Natl Acad. Sci. USA 111, E1629–E1638 (2014)

    Article  CAS  Google Scholar 

  16. Skovgaard, O., Bak, M., Lobner-Olesen, A. & Tommerup, N. Genome-wide detection of chromosomal rearrangements, indels, and mutations in circular chromosomes by short read sequencing. Genome Res. 21, 1388–1393 (2011)

    Article  CAS  Google Scholar 

  17. Neylon, C., Kralicek, A. V., Hill, T. M. & Dixon, N. E. Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex. Microbiol. Mol. Biol. Rev. 69, 501–526 (2005)

    Article  CAS  Google Scholar 

  18. Waldminghaus, T., Weigel, C. & Skarstad, K. Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome. Nucleic Acids Res. 40, 5465–5476 (2012)

    Article  CAS  Google Scholar 

  19. Breier, A. M., Weier, H. U. & Cozzarelli, N. R. Independence of replisomes in Escherichia coli chromosomal replication. Proc. Natl Acad. Sci. USA 102, 3942–3947 (2005)

    Article  ADS  CAS  Google Scholar 

  20. del Solar, G. et al. Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62, 434–464 (1998)

    Article  CAS  Google Scholar 

  21. Erdmann, S., Le Moine Bauer, S. & Garrett, R. A. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol. Microbiol. 91, 900–917 (2014)

    Article  CAS  Google Scholar 

  22. Smith, G. R. How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist’s view. Microbiol. Mol. Biol. Rev. 76, 217–228 (2012)

    Article  CAS  Google Scholar 

  23. Dillingham, M. S. & Kowalczykowski, S. C. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol. Mol. Biol. Rev. 72, 642–671 (2008)

    Article  CAS  Google Scholar 

  24. Kuzminov, A. Single-strand interruptions in replicating chromosomes cause double-strand breaks. Proc. Natl Acad. Sci. USA 98, 8241–8246 (2001)

    Article  ADS  CAS  Google Scholar 

  25. Michel, B. et al. Rescue of arrested replication forks by homologous recombination. Proc. Natl Acad. Sci. USA 98, 8181–8188 (2001)

    Article  ADS  CAS  Google Scholar 

  26. Shee, C. et al. Engineered proteins detect spontaneous DNA breakage in human and bacterial cells. eLife 2, e01222 (2013)

    Article  Google Scholar 

  27. Babu, M. et al. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair. Mol. Microbiol. 79, 484–502 (2011)

    Article  CAS  Google Scholar 

  28. Lin, L. Study of Bacteriophage T7 Gene 5. 9 and Gene 5. 5. PhD thesis, State Univ. New York. (1992)

  29. Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008)

    Article  ADS  CAS  Google Scholar 

  30. Guzman, L. M. et al. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995)

    Article  CAS  Google Scholar 

  31. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 1–11 (2006)

    Article  Google Scholar 

  32. Sharan, S. K. et al. Recombineering: a homologous recombination-based method of genetic engineering. Nature Protocols 4, 206–223 (2009)

    Article  CAS  Google Scholar 

  33. Datta, S., Costantino, N. & Court, D. L. A set of recombineering plasmids for gram-negative bacteria. Gene 379, 109–115 (2006)

    Article  CAS  Google Scholar 

  34. Waldminghaus, T., Weigel, C. & Skarstad, K. Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome. Nucleic Acids Res. 40, 5465–5476 (2012)

    Article  CAS  Google Scholar 

  35. Bidnenko, V., Ehrlich, S. D. & Michel, B. Replication fork collapse at replication terminator sequences. EMBO J. 21, 3898–3907 (2002)

    Article  CAS  Google Scholar 

  36. Svenningsen, S. L. et al. On the role of Cro in lambda prophage induction. Proc. Natl Acad. Sci. USA 102, 4465–4469 (2005)

    Article  ADS  CAS  Google Scholar 

  37. Yu, D. et al. An efficient recombination system for chromosome engineering in Escerichia coli. Proc. Natl Acad. Sci. USA 97, 5978–5983 (2000)

    Article  ADS  CAS  Google Scholar 

  38. Tischer, B. K. et al. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40, 191–197 (2006)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank N. Barkai, A. Tanay, E. Mick, S. Doron, A. Stern, T. Dagan and M. Shamir for discussion. We also thank the Skarstad group for providing the MG1655dnaC2 strain, the Michel group for providing the JJC1819 strain and D. Dar for assistance in Illumina sequencing. R.S. was supported, in part, by the Israel Science Foundation (personal grant 1303/12 and I-CORE grant 1796), the European Research Council Starting Grant programme (grant 260432), Human Frontier Science Program (grant RGP0011/2013), the Abisch-Frenkel foundation, the Pasteur-Weizmann Council, the Minerva Foundation, and by a Deutsch-Israelische Projektkooperation grant from the Deutsche Forschungsgemeinschaft. U.Q. was supported, in part, by the European Research Council Starting Grant programme (grant 336079), the Israel Science Foundation (grant 268/14) and the Israeli Ministry of Health (grant 9988-3). A.L. is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship.

Author information

Authors and Affiliations

Authors

Contributions

M.G., U.Q., A.L. and R.S. conceived and designed the research studies; M.G., I.Y., O.A., M.M., G.A. and R.E. performed the experiments; A.L., M.G., U.Q. and R.S. analysed data; A.L., M.G., U.Q. and R.S. wrote the manuscript.

Corresponding authors

Correspondence to Udi Qimron or Rotem Sorek.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Graphic overview of the procedure for characterizing the frequency and sequence of newly acquired spacers.

DNA from cultures of either E. coli K-12 (left) or E. coli BL21-AI (right) strains expressing Cas1–Cas2 from two different plasmids were used as templates for PCR. Round 1 was used to determine the frequency of spacer acquisition by comparing occurrences of expanded arrays to wild-type (WT) arrays. Round 2 amplified only the expanded arrays and, followed by deep sequencing, was used to determine the sequence, location and source of newly acquired spacers.

Extended Data Figure 2 PAMs and DNA content along the E. coli BL21-AI genome.

a, Distribution of PAM (AAG) sequences. Each data point represents the number of PAMs in a window of 10 kb. b, DNA content of a culture growing in log phase. Genomic DNA was extracted from E. coli BL21-AI cells carrying the pCas plasmid, grown at log phase, and was sequenced using the Illumina technology. The resulting reads were mapped to the sequenced E. coli BL21(DE3) genome (GenBank accession number NC_012947). Areas where few or no reads map to the genome represent regions that are present in the reference BL21(DE3) genome but are missing from the genome of the sequenced strain (BL21-AI).

Extended Data Figure 3 Distribution of newly acquired spacers on the genome during synchronized replication.

E. coli K-12ΔcasCdnaC2 cells were transferred from 39 °C (replication restrictive temperature) to 30 °C (replication permissive). Cas1–Cas2 were induced in these cells 30 min before the transfer to 30 °C and during the growth in 30 °C. Newly acquired spacers were sequenced at the given time points: a, following 20 min; b, following 40 min; c, following 60 min from replication initiation. The positions of the newly acquired spacers in windows of 100 kb are shown, and their fraction out of the total new spacers in the sample.

Extended Data Figure 4 A model explaining the preference for spacer acquisition near TerC compared with TerA in E. coli BL21-AI.

The DNA manipulation at the CRISPR region forms a replication fork stalling site, and leads to extensive spacer acquisition upstream of the CRISPR. While the clockwise fork is stalled at the CRISPR, the anticlockwise fork reaches the Ter region and is stalled at the respective Ter site, TerC, leading to extensive spacer acquisition upstream of TerC. Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the anticlockwise one (oriC to TerC), leading the forks to stall at TerC more often than at TerA.

Extended Data Figure 5 The protein product of T7 gene 5.9 inhibits spacer acquisition activity.

E. coli BL21-AI strains harbouring pBAD-Cas1+2 and pBAD33-gp5.9 (lane 1) or pBAD33 vector control (lane 2) were grown overnight in the presence of inducers (0.4% l-arabinose). Gel shows PCR products amplified from the indicated cultures using primers annealing to the leader and to the fifth spacer of the CRISPR array. Results represent one of three independent experiments.

Extended Data Figure 6 Distribution of protospacers across the plasmids.

a, Distribution across pCtrl-Chi; b, distribution across pChi plasmids. Circular representation of the 4.7 kb plasmid is presented, with the inserted 4-Chi cluster present at the top of the circle. Black bars indicate the number of PAM-derived spacers sequenced from each position; green bars represent non-PAM spacers. Scale bar, 100,000 spacers. Pooled protospacers from two replicates are presented for each panel.

Extended Data Table 1 Spacer acquisition in normal and perturbed conditions
Extended Data Table 2 Replication-dependent spacer acquisition
Extended Data Table 3 Involvement of the DNA repair machinery in spacer acquisition

Supplementary information

Supplementary Table 1

This file contains the data for Supplementary Table 1. (XLSX 657 kb)

Supplementary Table 2

This table contains the bacterial strains, plasmids and oligonucleotides used in this study. (XLSX 16 kb)

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Levy, A., Goren, M., Yosef, I. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015). https://doi.org/10.1038/nature14302

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