Single cell variability of CRISPR‐Cas interference and adaptation

• Published in: Molecular systems biology Vol. 18; no. 4; pp. e10680 - n/a
• Main Authors: McKenzie, Rebecca E, Keizer, Emma M, Vink, Jochem N A, van Lopik, Jasper, Büke, Ferhat, Kalkman, Vera, Fleck, Christian, Tans, Sander J, Brouns, Stan J J
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-04-2022; EMBO Press; John Wiley and Sons Inc; Springer Nature
• Subjects: Adaptation; Adaptation, Physiological - genetics; agent‐based simulations; Bacteria; Bacteriophages; Cell division; Clearances; CRISPR; CRISPR-Cas Systems - genetics; DNA - metabolism; E coli; EMBO23; Escherichia coli - genetics; Escherichia coli - metabolism; Gene expression; Heterogeneity; Interference; Mathematical models; Microfluidic devices; Microfluidics; Microscopy; Mutation; Phages; Plasmids; Population; Population studies; Priming; Proteins; single‐cell analysis; spacer acquisition; Stochasticity; Subpopulations; time‐lapse microscopy; type I CRISPR‐Cas; agent‐based simulations; spacer acquisition; single‐cell analysis; time‐lapse microscopy; type I CRISPR‐Cas; type I CRISPR-Cas; time-lapse microscopy; agent-based simulations; single-cell analysis
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.202110680

While CRISPR‐Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time‐lapse microscopy and mathematical modelling, we studied invader clearance in
Escherichia coli
across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell‐to‐cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR‐Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.
Synopsis
Time‐lapse microscopy combined with computational modeling reveals new insights into the single‐cell biology of CRISPR‐Cas defense during invader DNA clearance in
E. coli
.
CRISPR adaptation and interference over multiple generations can be tracked using time‐lapse microscopy.
Direct interference is fast and efficient, while priming shows large variations between cells.
Slower cell growth leads to earlier spacer acquisition within a population.
Mathematical modeling shows reduced Cascade binding affinity for the target drives variation between cells during priming.
Graphical Abstract
Time‐lapse microscopy combined with computational modeling reveals new insights into the single‐cell biology of CRISPR‐Cas defense during invader DNA clearance in
E
. 
coli
.