Staphylococcus aureus Cas9 (SaCas9)

RNA-guided DNA endonuclease from Staphylococcus aureus that cleaves double-stranded DNA at sites specified by a 21- to 23-nucleotide guide sequence, requiring an NNGRRT PAM immediately 3' of the target. At 1,053 amino acids it packages together with its guide RNA inside a single AAV vector, which SpCas9 does not.

Length: 3285 bp (1095 aa)

PAM: NNGRRT

Cut type: blunt

Effector domain: RuvC and HNH

Origin: Staphylococcus aureus type II CRISPR-Cas system

Characteristics

Cleaves both strands of the target three base pairs upstream of the PAM to produce a blunt double-strand break; the RuvC-like domain cuts the noncomplementary strand and the HNH domain cuts the strand complementary to the guide, while a separate PAM-interacting domain reads the NNGRRT motif. Guide spacers of 20 to 24 nucleotides give maximal activity, 18 and 19 nucleotides retain partial activity, and 17 nucleotides or shorter are inactive. At genomic sites carrying NGGRRT PAMs, which both SaCas9 and SpCas9 accept, the two enzymes produce indels at comparable rates. D10A inactivates RuvC and N580A inactivates HNH, each converting the 1,053-residue protein into a nickase; paired D10A nickases with guide offsets of 0 to 125 base pairs reach indel frequencies near 60% in HEK293 cells.

Applications: Validated for single-AAV in vivo editing. An AAV8 vector carrying SaCas9 and a Pcsk9 guide, injected intravenously in mice at 2 × 10¹¹ genome copies, produces over 40% indels in liver within one week, a ~95% fall in serum Pcsk9 and a ~40% fall in total cholesterol. AAV-delivered SaCas9 excises dystrophin exon 23 in mdx mice and restores dystrophin in skeletal and cardiac muscle. EDIT-101 pairs SaCas9 with two CEP290 guides in AAV5 for subretinal injection in Leber congenital amaurosis type 10.

Limitations: The NNGRRT PAM is more restrictive than the NGG PAM of SpCas9; the KKH variant (E782K/N968K/R1015H) relaxes recognition to NNNRRT and roughly doubles the number of targetable sites. Guide spacers of 17 nucleotides or shorter are inactive. Anti-SaCas9 antibodies occur in 78% of healthy adult donors and anti-SaCas9 T cells in 78%, a risk for in vivo therapeutic delivery.

Sequence

You must be signed in to view the full sequence.

References

  1. Ran et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature - Ran 2015 SaCas9
  2. Friedland et al. (2015). Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol - Friedland 2015 SaCas9
  3. Kleinstiver et al. (2015). Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nat Biotechnol - Kleinstiver 2015 SaCas9-KKH

Related collections

1 4