Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

Abstract

The adaptation phase of CRISPR–Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1–Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1–Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Whole-genome deep sequencing reveals off-target spacer integration events within the E. coli genome.
Fig. 2: Spacer-seq identifies hundreds of off-target spacer integration sites within the E. coli genome.
Fig. 3: Effects of genomic knockouts of IHF and the CRISPR1 locus on off-target spacer integration activity.
Fig. 4: Comparison of three different NCA sequences and their activity in target interference and primed acquisition.
Fig. 5: Evidence for native off-target spacer integrations.

References

  1. 1.

    Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

    CAS  Article  Google Scholar 

  3. 3.

    Wright, A. V., Nuñez, J. K. & Doudna, J. A. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164, 29–44 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Sternberg, S. H., Richter, H., Charpentier, E. & Qimron, U. Adaptation in CRISPR–Cas systems. Mol. Cell. 61, 797–808 (2016).

    CAS  Article  Google Scholar 

  5. 5.

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

    CAS  Article  Google Scholar 

  6. 6.

    Nuñez, J. K., Bai, L., Harrington, L. B., Hinder, T. L. & Doudna, J. A. CRISPR immunological memory requires a host factor for specificity. Mol. Cell. 62, 824–833 (2016).

    Article  Google Scholar 

  7. 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).

    CAS  Article  Google Scholar 

  8. 8.

    Levy, A. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Nuñez, J. K., Lee, A. S., Engelman, A. & Doudna, J. A. Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity. Nature 519, 193–198 (2015).

    Article  Google Scholar 

  10. 10.

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

    Article  Google Scholar 

  11. 11.

    Wright, A. V. et al. Structures of the CRISPR genome integration complex. Science 357, 1113–1118 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    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).

    CAS  Article  Google Scholar 

  13. 13.

    Shmakov, S. et al. Pervasive generation of oppositely oriented spacers during CRISPR adaptation. Nucleic Acids Res. 42, 5907–5916 (2014).

    CAS  Article  Google Scholar 

  14. 14.

    Wang, J. et al. Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR–Cas systems. Cell 163, 840–853 (2015).

    CAS  Article  Google Scholar 

  15. 15.

    Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. Molecular recordings by directed CRISPR spacer acquisition. Science 353, aaf1175 (2016).

    Article  Google Scholar 

  16. 16.

    McGinn, J. & Marraffini, L. A. CRISPR–Cas systems optimize their immune response by specifying the site of spacer integration. Mol. Cell. 64, 616–623 (2016).

    CAS  Article  Google Scholar 

  17. 17.

    Wright, A. V. & Doudna, J. A. Protecting genome integrity during CRISPR immune adaptation. Nat. Struct. Mol. Biol. 10, 876–883 (2016).

    Article  Google Scholar 

  18. 18.

    Wang, R., Ming, L., Gong, L., Hu, S. & Xiang, H. DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica. Nucleic Acids Res. 44, 4266–4277 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Goren, M. G. et al. Repeat size determination by two molecular rulers in the type I-E CRISPR array. Cell. Rep. 16, 2811–2818 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Rollie, C., Schneider, S., Brinkmann, A. S., Bolt, E. L. & White, M. F. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition. eLife 4, e08716 (2015).

    Article  Google Scholar 

  21. 21.

    Kunin, V., Sorek, R. & Hugenholtz, P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 8, R61 (2007).

    Article  Google Scholar 

  22. 22.

    Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacter. Nature 547, 345–349 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Marraffini, L. A. CRISPR–Cas immunity in prokaryotes. Nature 526, 55–61 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Datsenko, K. A., Pougach, K., Tikhonov, A., Wanner, B. L., Severinov, K. & Semenova, E. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat. Commun. 3, 945 (2012).

    Article  Google Scholar 

  25. 25.

    Kuznedelov, K. et al. Altered stoichiometry Escherichia coli cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation. Nucleic Acids Res. 44, 10849–10861 (2016).

    CAS  Article  Google Scholar 

  26. 26.

    Grissa, I., Vergnaud, G. & Pourcel, C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinforma. 8, 172 (2007).

    Article  Google Scholar 

  27. 27.

    Barros, M. P. S. et al. Dynamics of CRISPR loci in microevolutionary process of Yersinia pestis strains. PLoS ONE 9, e108353 (2014).

    Article  Google Scholar 

  28. 28.

    Eppinger, M. et al. Genome sequence of the deep-rooted Yersina pestis strain angola reveals new insights into the evolution and pangenome of the plague bacterium. J. Bacteriol. 192, 1685–1699 (2010).

    CAS  Article  Google Scholar 

  29. 29.

    Jaubert, C. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon. Open. Biol. 3, 130010 (2013).

    Article  Google Scholar 

  30. 30.

    Gudbergsdottir, S. et al. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers. Mol. Microbiol. 79, 35–49 (2011).

    CAS  Article  Google Scholar 

  31. 31.

    Reno, M. L., Held, N. L., Fields, C. J., Burke, P. V. & Whitaker, R. J. Biogeography of the Sulfolobus islandicus pan-genome. Proc Natl. Acad. Sci. USA 106, 8065–8610 (2009).

    Article  Google Scholar 

  32. 32.

    Jiang, Y. et al. Multigene editing in Escherichia coli genome via the CRISPR–Cas9 system. Appl. Environ. Microbiol. 81, 2506–2514 (2015).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank Ekaterina Semonova and Konstantin Severinov (Rutgers) for generously providing strain BW40114, and J. Doudna and L. Harrington (UCB) for generously providing BL21-AI IHF-knockout strains. The project was supported by grants from the National Human Genome Research Institute (5R01MH103910), the National Human Genome Research Institute (5RM1HG008525) and the Simons Foundation Autism Research Initiative (368485) to G.M.C.

Author information

Affiliations

Authors

Contributions

J.N. and S.L.S. conceived the study. J.N. designed the work, performed the experiments, analysed the data and wrote the manuscript with input from S.L.S. and G.M.C. S.L.S. and G.M.C. discussed the results and commented on the manuscript.

Corresponding author

Correspondence to George M. Church.

Ethics declarations

Competing interests

J.N., S.L.S. and G.M.C. are inventors on a provisional patent (62/490,901) filed by the President and Fellows of Harvard College that covers the work in this manuscript. A complete account of the financial interests of G.M.C. is listed at: http://arep.med.harvard.edu/gmc/tech.html.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Tables 1 and 2.

Life Sciences Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nivala, J., Shipman, S.L. & Church, G.M. Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration. Nat Microbiol 3, 310–318 (2018). https://doi.org/10.1038/s41564-017-0097-z

Download citation

Further reading