Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 systems are powerful genome editing tools but suffer from off-target effects.
• Staphylococcus aureus Cas9 (SaCas9) is favored for in vivo applications, yet lacks high-specificity variants.
• Improving SaCas9 specificity is crucial for safe and effective genome editing.

Purpose of the Study:

• To engineer SaCas9 variants with enhanced genome-wide specificity without compromising on-target efficiency.
• To evaluate the performance of engineered SaCas9 variants in human cells using unbiased and targeted methods.
• To assess the potential of these variants for in vivo applications and broader genome editing use.

Main Methods:

• Rational engineering of SaCas9 protein structure.
• Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq).
• Targeted deep sequencing to quantify on- and off-target editing events.
• Adeno-associated virus (AAV) vector delivery for in vitro studies.

Main Results:

• Engineered SaCas9-HF variant demonstrated dramatically improved genome-wide specificity.
• SaCas9-HF showed no detectable or minimal off-target activity at tested endogenous sites.
• On-target efficiencies of SaCas9-HF were comparable to wild-type SaCas9.
• Engineered KKH-HF variant also exhibited reduced off-target effects and improved editing ratios.
• Reduced off-target effects were observed with SaCas9-HF delivered via AAV vector.

Conclusions:

• Rationally engineered SaCas9 variants offer high genome-wide specificity in human cells.
• SaCas9-HF and KKH-HF provide safer alternatives to wild-type SaCas9 for precision genome editing.
• These variants are suitable for applications demanding exceptional genome-wide accuracy.