Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system

The adaptation against foreign nucleic acids by the CRISPR–Cas system (Clustered Regularly In-terspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of for-eign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mecha-nism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the inser-tion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a stag-gered way and is accompanied by joining of the re-sulting 5′-ends of the repeat strands with the 3′-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding cen-ter of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA