Engineered IscB-ωRNA system with improved base editing efficiency for disease correction via single AAV delivery in mice.

IF 7.5 1区生物学 Q1 CELL BIOLOGY

Cell reports Pub Date : 2024-11-13 DOI:10.1016/j.celrep.2024.114973

Ruochen Guo, Xiaozhi Sun, Feizuo Wang, Dingyi Han, Qiaoxia Yang, Hua Gao, Zhifang Li, Zhuang Shao, Jinqi Shi, Rongrong Yang, Xiaona Huo, Junda Yan, Guoling Li, Qingquan Xiao, Yuanhua Liu, Senfeng Zhang, Xinyu Liu, Yingsi Zhou, Leyun Wang, Chunyi Hu, Chunlong Xu

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Abstract

IscBs, as hypercompact ancestry proteins of Cas9 nuclease, are suitable for in vivo gene editing via single adeno-associated virus (AAV) delivery. Due to the low activity of natural IscBs in eukaryotic cells, recent studies have been focusing on improving OgeuIscB's gene editing efficiency via protein engineering. However, in vivo gene editing efficacy of IscBs for disease correction remained to be demonstrated. Here, we showed effective gene knockout and base editing in mouse embryos. To further improve IscB activity, we performed systematic engineering of IscB-associated ωRNA and identified a variant, ωRNA^∗-v2, with enhanced gene editing efficiency. Furthermore, our study demonstrated the efficacy of an engineered IscB-ωRNA system for robust gene knockout and base editing in vivo. Single AAV delivery of IscB-derived cytosine and adenine base editors achieved disease correction in a mouse model of tyrosinemia. Therefore, our results indicated the great potential of miniature IscBs for developing single-AAV-based gene editing therapeutics.

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工程化 IscB-ωRNA 系统提高了碱基编辑效率，可通过单次 AAV 给药对小鼠进行疾病矫正。

IscBs作为Cas9核酸酶的超小型祖先蛋白，适合通过单次腺相关病毒（AAV）传递进行体内基因编辑。由于天然 IscB 在真核细胞中的活性较低，近年来的研究主要集中在通过蛋白质工程提高 OgeuIscB 的基因编辑效率。然而，IscBs 在体内用于疾病矫治的基因编辑功效仍有待证实。在这里，我们在小鼠胚胎中展示了有效的基因敲除和碱基编辑。为了进一步提高IscB的活性，我们对IscB相关的ωRNA进行了系统工程改造，发现了一种基因编辑效率更高的变体ωRNA∗-v2。此外，我们的研究还证明了工程化 IscB-ωRNA 系统对体内基因敲除和碱基编辑的有效性。在酪氨酸血症小鼠模型中，单次AAV递送IscB衍生的胞嘧啶和腺嘌呤碱基编辑就能达到疾病矫正的效果。因此，我们的研究结果表明，微型 IscB 在开发基于单次 AAV 的基因编辑疗法方面具有巨大潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Cell reports CELL BIOLOGY-

CiteScore

13.80

自引率

1.10%

发文量

1305

审稿时长

77 days

期刊介绍： Cell Reports publishes high-quality research across the life sciences and focuses on new biological insight as its primary criterion for publication. The journal offers three primary article types: Reports, which are shorter single-point articles, research articles, which are longer and provide deeper mechanistic insights, and resources, which highlight significant technical advances or major informational datasets that contribute to biological advances. Reviews covering recent literature in emerging and active fields are also accepted. The Cell Reports Portfolio includes gold open-access journals that cover life, medical, and physical sciences, and its mission is to make cutting-edge research and methodologies available to a wide readership. The journal's professional in-house editors work closely with authors, reviewers, and the scientific advisory board, which consists of current and future leaders in their respective fields. The advisory board guides the scope, content, and quality of the journal, but editorial decisions are independently made by the in-house scientific editors of Cell Reports.