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

The 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. HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, 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 the hairpin-sgRNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an AAV vector and exhibited a 1.7∼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 γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of β-hemoglobinopathy. Together, eeCas9 and its derivates are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.