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Single cell variability of CRISPR-Cas interference and adaptation.

Rebecca E McKenzie1,2,3, Emma M Keizer4, Jochem N A Vink1,2

  • 1Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands.

Molecular Systems Biology
|April 25, 2022
PubMed
Summary

CRISPR-Cas immunity in bacteria shows fast, consistent clearance of invaders. However, mutations lead to variable responses due to primed adaptation, impacting bacterial defense strategies.

Keywords:
agent-based simulationssingle-cell analysisspacer acquisitiontime-lapse microscopytype I CRISPR-Cas

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • CRISPR-Cas systems provide adaptive immunity in bacteria against mobile genetic elements.
  • Previous studies focused on population-level CRISPR-Cas dynamics, leaving cellular-level temporal dynamics and variability unexplored.

Purpose of the Study:

  • To investigate the temporal dynamics and cell-to-cell variability of CRISPR-Cas defense mechanisms in individual bacterial cells.
  • To elucidate the mechanisms underlying heterogeneity in CRISPR-Cas mediated clearance of invading DNA.

Main Methods:

  • Utilized a microfluidic device for real-time observation of Escherichia coli populations.
  • Employed time-lapse microscopy to monitor invader clearance across multiple generations.
  • Integrated mathematical modeling to analyze clearance dynamics and identify sources of variability.

Main Results:

  • Observed rapid CRISPR interference with a narrow distribution of clearance times for non-mutated invaders.
  • Identified significant cell-to-cell variability in clearance times for PAM-mutated invaders, linked to primed CRISPR adaptation.
  • Demonstrated that faster growth, cell division, and higher Cascade levels favor clearance by interference, while slower growth favors priming.

Conclusions:

  • Primed CRISPR adaptation, driven by Cascade binding to mutated DNA, is a major source of heterogeneity in bacterial defense.
  • The stochastic nature of primed adaptation means only subpopulations can rapidly respond to new threats.
  • Cellular-level CRISPR-Cas dynamics and heterogeneity are critical for understanding bacterial competition and phage resistance.