Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase

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. Single-molecule FRET analysis of EfaCas1−Cas2 integrase establishes the kinetic framework for CRISPR spacer acquisition and shows that transcription effictively resolves the postsynaptic complex to ensure efficient and directional spacer integration.