Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Compact CRISPR-Cas9 systems packaged in adeno-associated virus (AAV) are promising for gene therapy.
• Current systems are limited by protospacer adjacent motif (PAM) requirements, restricting target scope.

Purpose of the Study:

• To optimize a small Cas9 ortholog (SpaCas9) for efficient in vivo genome editing.
• To expand the targeting repertoire of compact Cas9 systems by utilizing distinct PAMs.

Main Methods:

• Revisited and optimized Streptococcus pasteurianus Cas9 (SpaCas9) for genome editing.
• Developed Spa-cytosine base editor (CBE) and Spa-adenine base editor (ABE) systems.
• Engineered SpaCas9 variants by modifying the PAM-interacting domain.
• Utilized AAV delivery for in vivo gene editing in adult mice.

Main Results:

• SpaCas9 efficiently targets 5'-NNGYRA-3' PAMs, distinct from other small Cas9s.
• Spa-CBE and Spa-ABE achieved robust C-to-T and A-to-G conversions in vitro and in vivo.
• Engineered SpaCas9 variants further expanded targeting scope.
• Successfully generated mutant mice and demonstrated Pcsk9 gene editing in adult mouse liver, reducing cholesterol.

Conclusions:

• Optimized SpaCas9 broadens CRISPR-Cas9 toolsets for efficient gene modification.
• Distinct PAM recognition and small size make SpaCas9 suitable for therapeutic applications.
• SpaCas9 offers a versatile platform for in vivo gene editing and potential therapies.