Which Cas9 Variant Works Best for AAV Delivery?

For a single AAV vector, use SaCas9 (~3.2 kb) or a compact variant (Nme2Cas9, CjCas9, ~3.0 kb). If you need SpCas9's PAM flexibility, use a split-intein system across two vectors.

SpCas9 (~4.1 kb) plus a U6-guide cassette and regulatory elements exceeds the ~4.7 kb packaging limit in practice, leaving too little room for promoters and ITRs.

Replace SpCas9 with SaCas9 (~3.2 kb) and roughly 0.9 kb becomes available for a promoter, a second guide, or a tag.

• • PAM availability. SaCas9's longer PAM reduces the number of usable sites; check before committing to a target region.
• • Co-infection efficiency for split systems is the limiting factor in vivo; report the fraction of cells receiving both halves if measurable.
• • Base and prime editors do not fit in AAV in their standard forms. Dual-AAV and split-protein designs exist but remain technically demanding.
• • Immunity. Pre-existing immunity to Cas9 orthologues varies in human populations and can limit in vivo efficacy; this is a clinical rather than a bench consideration but worth knowing.

If the target cell type is easily transduced by lentivirus, or can be electroporated ex vivo, AAV's cargo constraint is an unnecessary complication. AAV earns its place when the target is post-mitotic tissue or when long-term episomal expression is needed in vivo.

EDITGENE advises on vector-format selection by cell type and target region, builds SaCas9 or compact-variant constructs where size is binding, and validates editing in the intended cells rather than in a surrogate line.

Tell us the target and the delivery route. Contact EDITGENE to request a project consultation or quote.

Packaging limits and construct sizes are approximate and depend on ITR configuration, promoter choice, and the specific variant; empirical titration is required.

Editing efficiency in the target tissue must be measured, not inferred from a packaging-validated construct.

• • Ran FA, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186–191.
• • Zetsche B, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163:759–771.
• • Edraki A, et al. A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Molecular Cell. 2019;73:714–726.
• • Wang D, et al. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery. 2019;18:358–378.