SpCas9 variant carrying the D10A substitution in the RuvC catalytic domain, which leaves only the HNH domain active so the enzyme nicks the DNA strand complementary to the guide RNA instead of cutting both strands. Used in pairs with offset guide RNAs for high-specificity editing, and as the DNA-binding nickase of cytosine and adenine base editors.
Length: 4200 bp (1400 aa)
PAM: NGG
Effector domain: HNH (RuvC inactivated by D10A)
Origin: Engineered from Streptococcus pyogenes Cas9 (D10A)
Characteristics
SpCas9 cuts the two DNA strands with two separate domains - HNH cleaves the strand complementary to the guide RNA and RuvC the noncomplementary strand - so the D10A substitution in RuvC leaves Cas9 D10A cleaving only the complementary strand. Target recognition is unchanged - a 20-nucleotide guide sequence and an NGG PAM immediately 3' of the target. Single nicks are repaired mainly by base excision repair and are poorly mutagenic; sequencing of 327 amplicons at the human EMX1 locus in HEK293FT cells detected no indels from Cas9 D10A alone, while homology-directed repair from a donor template proceeded at levels similar to wild-type SpCas9. Two Cas9 D10A complexes guided to opposite strands of one site nick cooperatively to form a staggered double-strand break.
Applications: Paired nicking with offset guide RNAs gives indel rates up to 40% at EMX1, DYRK1A, and GRIN2B in HEK293FT cells with 50- to 1,500-fold less off-target mutagenesis than wild-type SpCas9, ssODN-templated knock-in at EMX1, multiplexed deletions of 0.5-6 kb at DYRK1A, and Mecp2 disruption in mouse zygotes injected with Cas9 D10A mRNA. Cas9 D10A is also the nickase in the base editors BE3 and ABE7.10, where it nicks the strand opposite the deaminated base, biasing repair toward the edited strand.
Limitations: A single Cas9 D10A nick rarely yields indels, so gene disruption requires a second nickase on the opposite strand and therefore two NGG PAMs at a workable spacing. Only guide pairs generating 5' overhangs, with offsets from about -8 to +20 bp, give robust indel formation; pairs whose geometry gives a 3' overhang call for Cas9 H840A instead. Prime editors use Cas9 H840A, not D10A.
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