Staphylococcus aureus Cas9, KKH variant (SaCas9-KKH)

Engineered variant of Staphylococcus aureus Cas9 carrying three substitutions in the PAM-interacting domain - E782K, N968K, and R1015H - that relax the wild-type NNGRRT PAM to NNNRRT. Expands the number of targetable genomic sites two- to fourfold over wild-type SaCas9 while retaining its compact size for single-AAV delivery.

Length: 3162 bp (1054 aa)

PAM: NNNRRT

Cut type: blunt

Effector domain: RuvC and HNH

Origin: Engineered from Staphylococcus aureus Cas9 (E782K/N968K/R1015H)

Characteristics

Carries E782K, N968K, and R1015H substitutions in the PAM-interacting domain; all three are required together, since fewer than three fail to support robust editing. The substitutions relax the third position of the wild-type NNGRRT PAM from G to any nucleotide, giving NNNRRT recognition and increasing the number of targetable human genomic sites two- to fourfold. GUIDE-seq shows wild-type and KKH SaCas9 induce comparable numbers of off-target sites at their respective targets, so the PAM relaxation does not broadly increase off-target activity. At shared NNGRRT sites, wild-type SaCas9 retains somewhat higher on-target editing efficiency than KKH. Inherits SaCas9's 1,053-residue length, RuvC/HNH catalytic architecture, and blunt double-strand cleavage three base pairs upstream of the PAM.

Applications: Used for in vivo single-AAV genome editing. AAV9 delivery of KKH SaCas9 with a single guide corrects the DMD exon 51 reading frame in human iPSC-derived cardiomyocytes and restores dystrophin expression and grip strength in mdx mice. AAV-PHP.eB delivery of KKH SaCas9 targeting Myo6-C442Y rescues auditory brainstem responses in a semi-dominant mouse model of progressive hearing loss.

Limitations: Still requires an R at PAM positions 4 and 5 (NNNRRT), so targeting remains PAM-constrained rather than PAM-free. Wild-type SaCas9 retains higher on-target efficiency at shared NNGRRT sites, so it is preferable when a site already carries one. At 1,053 residues, KKH does not shrink SaCas9's own AAV packaging footprint any further.

References

  1. Ran et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature - Ran 2015 SaCas9
  2. Kleinstiver et al. (2015). Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nat Biotechnol - Kleinstiver 2015 SaCas9-KKH
  3. Zhang et al. (2021). A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation. Mol Ther Methods Clin Dev - Zhang 2021 SaCas9-KKH DMD

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