RNA-DNA杂交结合域拓宽了碱基编辑器的编辑窗口。

RNA-DNA hybrid binding domain broadens the editing window of base editors.

作者信息

Yang Yue, Fu Zihao, Deng Shengcheng, Wu Guanglan, Wang Chuanle, Luo Xiao, Kang Rui, Chen Yuxi, Peng Chengxiang, Zhang Pengfei, Cui Kaixin, Wan Fen, Wang Junhua, Zhou Qin, Chen Wei, Xiong Yuanyan, Ma Wenbin, Songyang Zhou, Liang Puping

机构信息

Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University & NHC Key Laboratory of Clinical Nephrology and Guangdong Provincial Key Laboratory of Nephrology, Guangzhou 510080, China; MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

出版信息

Mol Ther. 2025 Jun 14. doi: 10.1016/j.ymthe.2025.06.024.

DOI:10.1016/j.ymthe.2025.06.024

PMID:40518666

Abstract

Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.

摘要

腺嘌呤碱基编辑器（ABEs）和胞嘧啶碱基编辑器（CBEs）是精确基因组编辑的重要工具，但在原间隔相邻基序（PAM）附近位置的编辑活性有限，这限制了它们的应用。本研究探讨了将碱基编辑器与RNA-DNA杂交结合域（RHBDs）融合以增强其编辑活性的潜力。具体而言，将ABE8e与人类核糖核酸酶H1（RHBD1）的RHBD融合，可使PAM近端区域（A9-A15）的A到G编辑效率显著提高，最高可达3.5倍，同时减少脱靶胞嘧啶编辑。此外，RHBD1与ABEmax、BE4max和双碱基编辑器（eA&C-BEmax）兼容，可增强它们在PAM近端碱基处的编辑活性。值得注意的是，融合了RHBD1的BE4max在PAM近端区域（C9-C12）的C到T编辑效率提高了3.1倍。此外，我们证明融合了RHBD1的ABE8e可以有效编辑人类细胞中与疾病相关的单核苷酸变异（SNV），并在成年小鼠肝脏中验证了其有效性。这些发现凸显了RHBD在扩大编辑窗口方面的重要性，以及碱基编辑器在基因治疗和疾病建模中的适用性。