Development of an intein-mediated split–Cas9 system for gene therapy

TLDR

CRISPR/Cas9 can target any gene, but its large size (>4 kb) limits delivery by rAAV for gene therapy. The study aimed to create a split‑Cas9 system that overcomes rAAV packaging limits via split‑inteins. They fused each Cas9 fragment to split‑intein parts that reconstitute full Cas9 through trans‑splicing upon co‑expression, and incorporated a repair template on the same dual‑plasmid system. The split‑intein Cas9 exhibited wild‑type nuclease activity, enabled efficient HDR with a double‑nick strategy, and was successfully packaged and delivered by rAAV, demonstrating functional reconstitution in cells.

Abstract

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.