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

• Published in: Nature communications Vol. 15; no. 1; pp. 8140 - 11
• Main Authors: Zhao, Na, Zhou, Jian, Tao, Tianfu, Wang, Qi, Tang, Jie, Li, Dengluan, Gou, Shixue, Guan, Zhihong, Olajide, Joshua Seun, Lin, Jiejing, Wang, Shuo, Li, Xiaoping, Zhou, Jiankui, Gao, Zongliang, Wang, Gang
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17.09.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13/109; 45/23; 45/91; 631/181/2475; 631/337/4041/3196; 631/61/201/2110; Adenine; Adenine - metabolism; Animals; Bacteria; Cell size; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; Cytidine - genetics; Cytidine - metabolism; Cytidine deaminase; Cytidine Deaminase - genetics; Cytidine Deaminase - metabolism; Cytosine; Directed evolution; Directed Molecular Evolution - methods; Editing; Evolution; Gene Editing - methods; HEK293 Cells; Humanities and Social Sciences; Humans; Mammalian cells; Mammals; multidisciplinary; Science; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-52483-3

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. Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, the authors report a Continuous Directed Evolution system in Mammalian cells (CDEM) enabling the BE evolution in a full-length manner with Cas9 nickase.