Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands.
Kavli Institute of Nanoscience, Delft, The Netherlands.
Mol Syst Biol. 2022 Apr;18(4):e10680. 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.
虽然 CRISPR-Cas 防御机制已在群体水平上进行了研究，但它们在个体细胞中的时变动态和可变性仍不清楚。我们使用微流控装置、延时显微镜和数学模型，在多个世代中研究了大肠杆菌中外源物的清除。我们观察到 CRISPR 干扰速度很快，清除时间的分布很窄。相比之下，对于具有逃逸 PAM 突变的入侵者，我们发现细胞间存在很大的可变性，这种可变性源于有准备的 CRISPR 适应。更快的生长和细胞分裂以及更高水平的 Cascade 增加了通过干扰进行清除的机会，而较慢的生长则与通过引发进行清除的机会增加有关。我们的研究结果表明，Cascade 与突变入侵 DNA 的结合，而不是间隔序列的整合，是引发异质性的主要来源。引发的 CRISPR 适应的高度随机性意味着只有细菌的亚群能够快速响应入侵威胁。我们推测，细胞水平上的 CRISPR-Cas 动态和异质性对于理解细菌在与其他物种和噬菌体竞争中的策略至关重要。
