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Rebecca E McKenzie1, Cristóbal Almendros1, Jochem N A Vink1

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We developed CAPTURE, a sensitive method to detect CRISPR spacer acquisition events. This technique enhances the detection of new spacers from bacteriophages and plasmids, aiding in understanding microbial immunity.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • CRISPR-Cas systems provide adaptive immunity in prokaryotes by acquiring spacer sequences from foreign genetic elements.
  • Detecting de novo spacer acquisition is challenging due to its low frequency within cell populations.

Purpose of the Study:

  • To develop a highly sensitive method for detecting CRISPR spacer acquisition events.
  • To enable the study of spacer integration mechanisms and tracing in natural ecosystems.

Main Methods:

  • Developed CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis).
  • Employs two PCR steps with intermediate automated electrophoresis and size-selected DNA amplicon extraction.
  • This protocol removes unexpanded arrays, significantly increasing sensitivity.

Main Results:

  • CAPTURE achieves high sensitivity, detecting spacer acquisition in as few as 1 in 10^5 cells.
  • The method is 1,000 times more sensitive than alternative PCR protocols.
  • Demonstrates versatility across native CRISPR systems.

Conclusions:

  • CAPTURE is a rapid (1-day) and adaptable protocol for sensitive detection of CRISPR spacer acquisition.
  • Facilitates research into the mechanisms of spacer integration.
  • Enables more sensitive tracing of integration events in diverse environments.