Abstract
The adaptation against foreign nucleic acids by the CRISPR–Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. 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 insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5′-ends of the repeat strands with the 3′-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center 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.
Highlights
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive prokaryotic defense system against foreign genetic elements, like phages or plasmids [1,2,3]
To study the mechanism of spacer integration into the CRISPR array in vivo, we adopted the assay developed by Yosef et al [23]
Induction of the plasmid-borne cas1–cas2 genes followed by growth of the cells for 18 h initiated the integration of new spacer DNA into the chromosomal CRISPR array (Supplementary Figure spacer 1 (S1))
Summary
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive prokaryotic defense system against foreign genetic elements, like phages or plasmids [1,2,3]. Induction of the plasmid-borne cas1–cas2 genes followed by growth of the cells for 18 h initiated the integration of new spacer DNA into the chromosomal CRISPR array (Supplementary Figure S1).
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