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

作者：Yin, Shuming; Zhang, Mei; Liu, Yang; Sun, Xiaoyue; Guan, Yuting; Chen, Xi; Yang, Lei; Huo, Yanan; Yang, Jing; Zhang, Xiaohui; Han, Honghui; Zhang, Jiqin; Xiao, Min -Min; Liu, Mingyao; Hu, Jiazhi^*; Wang, Liren^*; Li, Dali^*

来源：Molecular Therapy, 2023, 31(3): 744-759.

DOI：10.1016/j.ymthe.2022.11.014

• RNA
• NUCLEASES
• COMPLEX

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.

• 单位
  北京大学