Visualizing the interplay of Cas1–Cas2 with DNA replication-repair that creates CRISPR–Cas immunity

Abstract

Prokaryotic CRISPR–Cas systems rely on the Cas1–Cas2 protein complex to capture new DNA from mobile genetic elements (MGEs), to form immunological memory that defends against the MGEs. However, the mechanisms by which Cas1–Cas2 locates suitable DNA substrates inside cells remain unclear, limiting our understanding of how CRISPR–Cas immunity arises de novo. We directly visualized functional, DNA-bound Cas1–Cas2 complexes in bacteria, revealing the processes that license Cas1–Cas2 to capture DNA. Visible DNA-bound Cas1–Cas2 complexes formed only when replisomes are actively advancing, accumulating at post-replicative DNA gaps behind replication forks—structures arising during normal genome duplication, which are normally repaired by homologous recombination. Replication stress, which increases replicative DNA gap frequency, enhanced visible Cas1–Cas2 DNA binding. DNA capture by Cas1–Cas2 was strongly stimulated in cells lacking the RecFOR complex, which normally directs DNA gaps to repair. The RecBCD recombination initiator complex was essential for DNA capture by Cas1–Cas2 in these cells. The findings support a model in which naïve CRISPR–Cas adaptation is licensed by abundant replication-dependent DNA repair intermediates, prior to their repair by recombination. This identifies the mechanism co-ordinating Cas1–Cas2 with essential DNA replication and repair processes that all cells need, including when they are hijacked to replicate parasitic MGEs.