HuidaGene Therapeutics Co. Ltd., 6th Floor, Unit 3, Building 5, No. 160 Basheng Road, Pudong New Area, Shanghai, 200131, China.
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Science, 320 Yueyang Road, Shanghai, 200031, China.
Adv Sci (Weinh). 2023 Jun;10(17):e2206813. doi: 10.1002/advs.202206813. Epub 2023 Apr 25.
Catalytically inactive CRISPR-Cas13 (dCas13)-based base editors can achieve the conversion of adenine-to-inosine (A-to-I) or cytidine-to-uridine (C-to-U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199-amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre-crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A-to-I and C-to-U base editing with low transcriptome off-target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases.
基于无催化活性的 CRISPR-Cas13（dCas13）的碱基编辑器可以在 RNA 水平上实现腺嘌呤向肌苷（A-to-I）或胞嘧啶向尿嘧啶（C-to-U）的转换，然而，dCas13 蛋白的体积大限制了其在体内的应用。本研究报道了一种紧凑且高效的 RNA 碱基编辑器（ceRBE），具有较高的体内编辑效率。该 ceRBE 用源自参与前 crRNA 加工的 Class 1 CRISPR 家族的 199 个氨基酸的 EcCas6e 蛋白替代了较大的 dCas13 蛋白，并针对毒性和编辑效率进行了优化。ceRBE 在 HEK293T 细胞中能够高效实现 A-to-I 和 C-to-U 碱基编辑，同时转录组脱靶率较低。在 Duchenne 肌营养不良症（DMD）的人源化小鼠模型中，通过 AAV 递送后，ceRBE 也能有效修复 DMD Q1392X 突变（68.3±10.1%），实现基因产物表达的恢复。该研究支持紧凑且高效的 ceRBE 具有治疗遗传疾病的巨大潜力。
