1. ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems
- Author
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Zhen Ouyang, Longquan Quan, Hui Shi, Nan Li, Xia Wang, Z. Zhuang, Jiaowei Wang, Fangbing Chen, Xiaozhu Zhao, Meng Lian, Liangxue Lai, Yanhui Liang, Jingke Xie, Han Wu, Xingyun Huang, Shixue Gou, Quanjun Zhang, Qin Jin, Weikai Ge, Yinghua Ye, and Kepin Wang
- Subjects
Physiology ,Sus scrofa ,Locus (genetics) ,Plant Science ,Computational biology ,Biology ,Mammalian systems ,General Biochemistry, Genetics and Molecular Biology ,Cytosine ,Mice ,chemistry.chemical_compound ,Fetus ,Structural Biology ,Simultaneous C-to-T and A-to-G conversions ,Animals ,Humans ,Point Mutation ,lcsh:QH301-705.5 ,Ecology, Evolution, Behavior and Systematics ,Subgenomic mRNA ,Gene Editing ,Cas9 ,Adenine ,Point mutation ,Methodology Article ,HEK 293 cells ,Cell Biology ,Cytidine deaminase ,Fibroblasts ,Embryo, Mammalian ,Adenine and cytosine base editor (ACBE) ,HEK293 Cells ,chemistry ,lcsh:Biology (General) ,General Agricultural and Biological Sciences ,Linker ,Developmental Biology ,Biotechnology - Abstract
BackgroundMany favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site.ResultsIn this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system.ConclusionThe newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.
- Published
- 2020