Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1–Cas2 integrase

Abstract

Edited by Patrick SungSite-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro. However, this nonspecific integration activity is rare in vivo. Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mM spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system

“…During recurring infections, swift expression of the latest spacers ensures a productive fight by a rapid and robust immune response (54). In conjunction with Cas1-2, various conserved DNA motifs present in the leader and repeat regions mandate the fidelity of prespacer integration (16,17,54,(57)(58)(59)(60)(61)(62)(63)(64)(65). In E. coli (type I-E), upon interaction with these conserved regions, a sequence-specific genome architectural protein termed integration host factor (IHF) restructures the leader and generates a docking site for Cas1-2 integrase at the leaderrepeat junction (16,17,66).…”