Investigating single-cell behaviour during CRISPR-Cas defence using time-lapse microscopy Variation in target plasmid clearance times is much larger when CRISPR adaptation is required Details of CRISPR-dependent plasmid loss and subsequent fluorescence decay Growth rate and interdivision times have an influence on direct interference and priming Cellular Cascade concentrations influence the direct interference response Cascade copy number determination Correlation of RFP levels between cells related as sisters, cousins and second cousins Schematic representation of the agent-based simulation framework Results from the stochastic agent-based model of CRISPR adaptation and interference Distribution of primed adaptation and primed interference time for high and low variability in Cascade concentration Slower-growing cells have higher Cascade concentrations Single cell variability of CRISPR-Cas interference and adaptation

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.


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.


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