Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.

中文翻译：

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通过Cas1-Cas2整合酶实时观察CRISPR间隔子。

Cas1整合酶与Cas2结合以将短的DNA片段插入CRISPR阵列，从而在原核生物中建立核酸记忆。在这里，我们将单分子FRET方法应用于粪肠球菌（Efa）Cas1-Cas2系统，以建立描述靶标搜索，整合和突触后事件的动力学框架。EfaCas1-Cas2本身无法在CRISPR阵列中找到CRISPR重复序列；它仅在加载预分隔符后才这样做。与重复序列相邻的前导序列进一步稳定了EfaCas1-Cas2接触，实现了前导侧整合和随后的间隔子侧整合。所得的突触后复合物（PSC）具有令人惊讶的平均寿命短。值得注意的是，转录有效地解决了PSC，并且我们预测这是一种保守的机制，可确保在许多CRISPR系统中高效且有方向性的间隔子整合。总体而言，我们的研究提供了一个完整的间隔物捕获模型，可用于基于DNA的信息存储和细胞谱系追踪技术。