Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
Nat Struct Mol Biol. 2020 May;27(5):489-499. doi: 10.1038/s41594-020-0415-7. Epub 2020 May 4.
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.
Cas1 整合酶与 Cas2 结合，将短 DNA 片段插入到 CRISPR 阵列中，在原核生物中建立核酸记忆。在这里，我们应用单分子 FRET 方法研究了粪肠球菌（Efa）Cas1-Cas2 系统，建立了一个描述靶标搜索、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；只有在前导序列加载后才能找到。重复序列旁边的前导序列进一步稳定了 EfaCas1-Cas2 的接触，从而能够进行先导侧整合和随后的间隔子侧整合。由此产生的突触后复合物（PSC）具有惊人的短平均寿命。值得注意的是，转录有效地解决了 PSC，我们预测这是一种保守的机制，可确保许多 CRISPR 系统中有效且定向的间隔子整合。总体而言，我们的研究提供了一个完整的间隔子获取模型，可用于基于 DNA 的信息存储和细胞谱系追踪技术。
