Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation - PubMed
3 hours ago
- #Genome Editing
- #Gene Therapy
- #CRISPR-Cas9
- Developed eSaCas9-NNG, a compact Staphylococcus aureus Cas9 variant with a relaxed NNG PAM requirement, increasing its targeting range.
- eSaCas9-NNG maintains high target fidelity, overcoming the common trade-off between expanded targeting and reduced specificity in Cas9 variants.
- Demonstrated efficient gene editing (indels and base conversions) at NNG PAM sites in human cells and mice, with editing efficiency comparable to tools like SpRY and SpG.
- Off-target activity of eSaCas9-NNG is reduced compared to other PAM-relaxed nucleases, enhancing its safety for genome editing.
- Cryo-EM structures of eSaCas9-NNG in five functional states provide mechanistic insights into relaxed PAM recognition, improved specificity, and nuclease activation.
- Potential application as a versatile genome editing tool for in vivo gene therapy, supported by a patent application from the University of Tokyo and MODALIS Corporation.