Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity

Ran et al. developed a double-nicking strategy using paired SpCas9 D10A nickase mutants (Cas9n) directed by offset guide RNAs targeting opposite DNA strands. Because individual nicks are repaired by high-fidelity base excision repair, simultaneous cooperative nicking generates targeted double-strand breaks requiring both guides to bind. This effectively doubles the recognized target sequence length relative to wild-type Cas9.

Paired nickase editing reduced off-target mutagenesis by 50- to 1,500-fold across multiple genomic loci in HEK293FT cells compared to wild-type SpCas9, as measured by SURVEYOR and deep sequencing assays. Optimal double-nicking efficiency required sgRNA pairs with positive offsets of 0–100 bp generating 5′ overhangs, while on-target indel rates remained comparable to wild-type Cas9.

Ran et al. applied double nicking to achieve high-specificity gene knockout in mouse zygotes, targeting Tet1 and Tet3 loci and confirming homozygous disruption in offspring. The strategy also supported homology-directed repair for precise allelic modification, demonstrating broad applicability for both knockout and knock-in genome editing applications requiring high specificity.