Abstract
CRISPR–Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 105 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. CAPTURE is a straightforward method that requires only 1 d to enable the detection of spacer acquisition in all native CRISPR systems and facilitate studies aimed both at unraveling the mechanism of spacer integration and more sensitive tracing of integration events in natural ecosystems.
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References
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Jackson, S. A. et al. CRISPR-Cas: adapting to change. Science 356, eaal5056 (2017).
Brouns, S. J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).
Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).
Heler, R. et al. Cas9 specifies functional viral targets during CRISPR–Cas adaptation. Nature 519, 199–202 (2015).
Hynes, A. P., Villion, M. & Moineau, S. Adaptation in bacterial CRISPR-Cas immunity can be driven by defective phages. Nat. Commun. 5, 4399 (2014).
Staals, R. H. J. et al. Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR–Cas system. Nat. Commun. 7, 12853 (2016).
Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat. Commun. 3, 945 (2012).
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).
Swarts, D. C., Mosterd, C., van Passel, M. W. J. & Brouns, S. J. J. CRISPR interference directs strand specific spacer acquisition. PLoS One 7, e35888 (2012).
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).
Silas, S. et al. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein. Science 351, aad4234 (2016).
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).
Amlinger, L., Hoekzema, M., Wagner, E. G. H., Koskiniemi, S. & Lundgren, M. Fluorescent CRISPR adaptation reporter for rapid quantification of spacer acquisition. Sci. Rep. 7, 10392 (2017).
Díez-Villaseñor, C., Guzmán, N. M., Almendros, C., García-Martínez, J. & Mojica, F. J. M. 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).
Schmidt, F., Cherepkova, M. Y. & Platt, R. J. Transcriptional recording by CRISPR spacer acquisition from RNA. Nature 562, 380–385 (2018).
Kieper, S. N. et al. Cas4 facilitates PAM-compatible spacer selection during CRISPR adaptation. Cell Rep. 22, 3377–3384 (2018).
Edwards, R. A., McNair, K., Faust, K., Raes, J. & Dutilh, B. E. Computational approaches to predict bacteriophage–host relationships. FEMS Microbiol. Rev. 40, 258–272 (2016).
Snyder, J. C., Bateson, M. M., Lavin, M. & Young, M. J. Use of cellular CRISPR (clusters of regularly interspaced short palindromic repeats) spacer-based microarrays for detection of viruses in environmental samples. Appl. Environ. Microbiol. 76, 7251–7258 (2010).
Shariat, N. & Dudley, E. G. CRISPRs: molecular signatures used for pathogen subtyping. Appl. Environ. Microbiol. 80, 430–439 (2014).
Modell, J. W., Jiang, W. & Marraffini, L. A. CRISPR–Cas systems exploit viral DNA injection to establish and maintain adaptive immunity. Nature 544, 101–104 (2017).
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).
Biswas, A., Gagnon, J. N., Brouns, S. J. J., Fineran, P. C. & Brown, C. M. CRISPRTarget: bioinformatic prediction and analysis of crRNA targets. RNA Biol. 10, 817–827 (2013).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Biswas, A., Staals, R. H. J., Morales, S. E., Fineran, P. C. & Brown, C. M. CRISPRDetect: a flexible algorithm to define CRISPR arrays. BMC Genomics 17, 356 (2016).
Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Díez-Villaseñor, C., Almendros, C., García-Martínez, J. & Mojica, F. J. M. Diversity of CRISPR loci in Escherichia coli. Microbiology 156, 1351–1361 (2010).
Acknowledgements
S.J.J.B. is supported by European Research Council (ERC) StG grant 639707, NWO Vidi grant 864.11.005 and a TU Delft startup grant. This work was supported by the Netherlands Organisation for Scientific Research (NWO/OCW), as part of the Frontiers of Nanoscience (NanoFront) program. The authors thank members of the Brouns lab for discussions on the manuscript and feedback.
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R.E.M., C.A., J.N.A.V. and S.J.J.B. conceived and designed the experiments; R.E.M., C.A. and J.N.A.V. performed the experiments; R.E.M., C.A., J.N.A.V. and S.J.J.B. analyzed the data; R.E.M. and S.J.J.B. wrote the paper with input from all other authors.
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Kieper, S. N. et al. Cell Rep. 22, 3377–3384 (2018): https://doi.org/10.1016/j.celrep.2018.02.103
Integrated supplementary information
Supplementary Figure 1 Sequence view of primer sets designed for CAPTURE.
The double-stranded DNA sequence of the array is shown here, including the leader sequence (black), repeat sequences (gray) and the sequence of spacer 1 (blue). Primer sets designed for the type I-E CRISPR system of E. coli K12 are indicated with black boxes and directional arrows.
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Supplementary Figure 1 and Supplementary Table 1
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McKenzie, R.E., Almendros, C., Vink, J.N.A. et al. Using CAPTURE to detect spacer acquisition in native CRISPR arrays. Nat Protoc 14, 976–990 (2019). https://doi.org/10.1038/s41596-018-0123-5
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DOI: https://doi.org/10.1038/s41596-018-0123-5
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