In vivo genome editing using Staphylococcus aureus Cas9

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• The Cas9 enzyme is a powerful tool for genome editing.
• The large size of Streptococcus pyogenes Cas9 (SpCas9) restricts its use with adeno-associated virus (AAV) delivery systems.
• Smaller Cas9 variants are needed for efficient AAV-mediated gene editing.

Purpose of the Study:

• To characterize smaller Cas9 orthologues for genome editing.
• To evaluate the efficiency and specificity of Staphylococcus aureus Cas9 (SaCas9) for in vivo gene editing.
• To demonstrate the therapeutic potential of SaCas9 using AAV delivery.

Main Methods:

• Characterization of six smaller Cas9 orthologues.
• In vivo genome editing in mouse liver targeting the Pcsk9 gene using SaCas9 delivered via AAV.
• Assessment of gene modification efficiency and reduction in serum Pcsk9 and cholesterol levels.
• Genome-wide specificity analysis using BLESS (Binding and Ligation-based Exonuclease Sequencing).

Main Results:

• Staphylococcus aureus Cas9 (SaCas9) demonstrated genome editing efficiencies comparable to SpCas9.
• SaCas9 is over 1 kilobase shorter than SpCas9, facilitating AAV packaging.
• In vivo SaCas9 delivery resulted in >40% Pcsk9 gene modification in mouse liver within one week.
• Significant reductions in serum Pcsk9 and total cholesterol levels were observed post-editing.
• SaCas9 exhibited efficient and specific genome-wide targeting in vivo.

Conclusions:

• SaCas9 is a viable, smaller alternative to SpCas9 for genome editing applications.
• AAV-packaged SaCas9 enables efficient in vivo gene editing with therapeutic potential.
• SaCas9-mediated editing offers a promising strategy for managing conditions like hypercholesterolemia.