2. Academic Validation
  3. A base editor facilitates simultaneous purine and pyrimidine substitutions for ex vivo and in vivo mutagenesis screens

A base editor facilitates simultaneous purine and pyrimidine substitutions for ex vivo and in vivo mutagenesis screens

  • Cell Chem Biol. 2025 Sep 18;32(9):1183-1196.e5. doi: 10.1016/j.chembiol.2025.08.003.
Zhiyong He 1,  Yuyang Xie 1,  Honglin Huang 1,  Zhaoyu Zhang 2,  Shenjiong Feng 1,  Runda Xu 3,  Xuancheng Chen 3,  Fei Gao 1,  Pan Li 4,  Ming Zhu 5,  Sen Wu 6,  Xuguang Du 7
Affiliations

Affiliations

  • 1 Frontiers Science Center for Molecular Design Breeding, State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, China.
  • 2 Sanya Institute of China Agricultural University, Sanya, China.
  • 3 Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
  • 4 Xianghu Laboratory, Hangzhou, China.
  • 5 Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. Electronic address: mzhu@bio.ecnu.edu.cn.
  • 6 Frontiers Science Center for Molecular Design Breeding, State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, China; Sanya Institute of China Agricultural University, Sanya, China. Electronic address: swu@cau.edu.cn.
  • 7 Frontiers Science Center for Molecular Design Breeding, State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, China; Sanya Institute of China Agricultural University, Sanya, China. Electronic address: xuguangdu@cau.edu.cn.
PMID: 40912250 DOI: 10.1016/j.chembiol.2025.08.003
Abstract

Genetic mutations are closely linked to human diseases, yet the relationship between many mutations and their corresponding phenotypes remains poorly understood. Furthermore, tools to study the connection between nucleotide variations and phenotypes are limited. To address this issue, we developed ACGBEmax by fusing the dual-functional deaminase, engineered N-methylpurine DNA glycosylase, and evolved SOS response associated peptidase domain with nCas9(D10A). ACGBEmax enables the precise conversion of A, C, and G to other Bases in mammalian cells, thereby generating an extensive range of base mutations types. We used ACGBEmax to generate HPRT variants, identifying mutations conferring resistance to 6-thioguanine. Additionally, we performed in situ mutagenesis of Ctnnb1 in mouse liver, identifying both known and potential oncogenic mutations. Our results prove that ACGBEmax is a powerful tool for generating a wide spectrum of mutation types at specific gene loci, highlighting its significant potential for applications in functional screening and the directed evolution of protein variants.

Keywords

CRISPR; DNA deaminase; DNA glycosylase; base editors; protein mutation screening.

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