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

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.