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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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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key reference using this protocol
Kieper, S. N. et al. Cell Rep. 22, 3377–3384 (2018): https://doi.org/10.1016/j.celrep.2018.02.103
Integrated supplementary information
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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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
Biochemical Society Transactions (2020)
Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems
FEMS Microbiology Letters (2019)
Nucleic Acids Research (2019)