Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of g-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate g-globin expression for the treatment of b-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.

Automated summary

Editing efficiency is crucial for CRISPR-based gene therapies, and this study demonstrates that fusing an HMG-D domain to the N terminus of SpCas9 significantly enhances editing efficiency. The HMG-D domain also improves the activity of other Cas9 orthologs and Cas9-based epigenetic regulators. It is found that eeCas9 exhibits comparable off-target effects with Cas9, but its specificity can be increased through different methods. In vivo and ex vivo experiments show that eeCas9 and its derivatives have higher editing activity and therapeutic efficacy in mice and human cells.