Nickase

Nickase is an enzyme that cleaves only one strand of double-stranded DNA, producing a single-strand nick rather than a double-strand break. In molecular biology and genome engineering, nickases are engineered or natural nucleases designed to nick DNA at specific sequences or structures. A prominent example is the Cas9 nickase, produced by mutating one of the two catalytic nuclease domains of the Cas9 endonuclease. Cas9 nickases generate single-stranded breaks on the DNA strand complementary to the guide RNA.

To create a double-strand break with greater specificity, two nickases can be used with two guide RNAs targeting nearby sites on opposite DNA strands. The two nicks together mimic a DSB, but off-target activity is often reduced because simultaneous off-target nicking at two nearby sites is less likely. This paired nicking strategy is widely used to improve targeting accuracy in CRISPR-based editing.

Nickases are also produced from naturally occurring or engineered nicking endonucleases that cut only one DNA strand, such as Nb.BbvCI or Nt.BbvCI, derived from BbvI restriction enzymes. These enzymes are used in molecular biology applications including DNA assembly, mapping, and single-strand nicking techniques.

Applications of nickases include genome editing with enhanced specificity, base editing when combined with deaminases, and certain diagnostic or nanotechnological tools. Limitations include the need for careful guide RNA design, potential incomplete nick repair, and residual off-target nicking in some contexts.