Cas9n

Cas9 nickase, or Cas9n, refers to nickase variants of the CRISPR-associated Cas9 enzyme that cleave only a single strand of DNA rather than producing a double-strand break. These variants typically carry a mutation in one nuclease domain, most commonly D10A in the RuvC domain or H840A in the HNH domain, which disables one catalytic site and yields a nick instead of a DSB.

The nicking activity can be leveraged in two main ways. A single nick is usually repaired by base excision repair pathways and produces relatively few indels. When two nickases guided by two RNA guides target opposite strands near each other, the two nicks create a staggered double-strand break at the intended locus, enabling genome editing with substantially higher target specificity than a single Cas9 cut. This two-nickase, paired-guide approach reduces off-target DSBs because two independent recognition events are required.

Cas9n is used in standard editing workflows where higher specificity is desirable, and it is also a component of certain base-editing systems in which a nickase is fused to a cytidine or adenosine deaminase to bias repair outcomes. In addition to editing, nickases have been explored for enabling precise homology-directed repair, often in conjunction with donor templates, while limiting unintended mutations.

Advantages of Cas9n include reduced off-target cleavage and compatibility with existing CRISPR workflows. Limitations include potentially lower editing efficiency and the need to design and deliver two guides, which increases complexity. Cas9n remains a widely used tool in genome engineering, particularly when specificity is a priority.