Rapid CRISPR-Cas9 target-strand nicking can provide phage resistance by reducing DNA abundance.

Cas9 is an RNA-guided immune endonuclease that provides bacterial defense against bacteriophages. Cas9 relies on divalent metal ions for cleavage catalysis by two domains, HNH and RuvC, and to facilitate conformational changes that are required for cleavage activation. While Cas9 typically produces double-strand breaks (DSBs) in DNA targets, we observed that reduced, physiologically relevant Mg2+ concentrations can result in a slow rate of non-target strand cleavage by RuvC. This raised the question of whether rapid target-strand nicking by the Cas9 HNH domain is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only the HNH or RuvC domain is catalytically active. We find that nicking by HNH, but not RuvC, can be sufficient to provide immunity. Target-strand nicking prevents phage DNA accumulation and can reduce the susceptibility of Cas9 to viral escape. Cleavage by RuvC is strongly impaired in the presence of other biomolecules that can compete for binding of free Mg2+, preventing formation of a DSB. Overall, our results suggest that HNH cleavage may occur more rapidly than RuvC cleavage under physiological conditions, resulting in an initial target-strand nick that may be sufficient to provide CRISPR-mediated immunity.