Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells

IF 14.7 1区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES Nature Communications Pub Date : 2024-09-17 DOI:10.1038/s41467-024-52483-3

Na Zhao, Jian Zhou, Tianfu Tao, Qi Wang, Jie Tang, Dengluan Li, Shixue Gou, Zhihong Guan, Joshua Seun Olajide, Jiejing Lin, Shuo Wang, Xiaoping Li, Jiankui Zhou, Zongliang Gao, Gang Wang

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Abstract

Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase. We harness CDEM to evolve the deaminases of cytosine base editor BE3 and adenine base editors, ABEmax and ABE8e. The evolved cytidine deaminase variants on BE4 architecture show not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity. The evolved ABEmax and ABE8e variants exhibit narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate that CDEM is a simple but powerful approach to continuously evolve BEs without size restriction in the mammalian environment, which is advantageous over continuous directed evolution system in bacteria cells.

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通过哺乳动物细胞中的持续定向进化系统，进化出高精度的胞苷和腺嘌呤碱基编辑器，并将脱靶活性降至最低

碱基编辑器（BE）的持续定向进化在细菌细胞中已经取得了成功，但在哺乳动物细胞中尚未成功。在这里，我们报告了哺乳动物细胞连续定向进化系统（CDEM）的开发情况。CDEM 利用 Cas9 标记酶以全长方式实现了 BE 的进化。我们利用 CDEM 进化了胞嘧啶碱基编辑器 BE3 和腺嘌呤碱基编辑器 ABEmax 和 ABE8e 的脱氨酶。在 BE4 架构上进化出的胞苷脱氨酶变体不仅缩小了编辑窗口，还提高了编辑纯度和低脱靶活性，同时不影响靶上活性。进化的 ABEmax 和 ABE8e 变体在不同程度上表现出编辑窗口变窄或移动，以及较低的脱靶效应。研究结果表明，CDEM是一种简单而强大的方法，可以在哺乳动物环境中不受大小限制地持续进化BE，这比细菌细胞中的持续定向进化系统更有优势。

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来源期刊

Nature Communications Biological Science Disciplines-

CiteScore

24.90

自引率

2.40%

发文量

6928

审稿时长

3.7 months

期刊介绍： Nature Communications, an open-access journal, publishes high-quality research spanning all areas of the natural sciences. Papers featured in the journal showcase significant advances relevant to specialists in each respective field. With a 2-year impact factor of 16.6 (2022) and a median time of 8 days from submission to the first editorial decision, Nature Communications is committed to rapid dissemination of research findings. As a multidisciplinary journal, it welcomes contributions from biological, health, physical, chemical, Earth, social, mathematical, applied, and engineering sciences, aiming to highlight important breakthroughs within each domain.