ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems
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METHODOLOGY ARTICLE
Open Access
ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems Jingke Xie1,2,3†, Xingyun Huang1,3†, Xia Wang1,4, Shixue Gou1,3, Yanhui Liang1,3, Fangbing Chen1,3, Nan Li1,2,3, Zhen Ouyang1,2,4, Quanjun Zhang1,2,4, Weikai Ge1,2,3, Qin Jin1,2,3, Hui Shi1,3, Zhenpeng Zhuang1,3, Xiaozhu Zhao1,3, Meng Lian1,5, Jiaowei Wang1,3, Yinghua Ye1,2,4, Longquan Quan1,2,4, Han Wu1,2,4, Kepin Wang1,2,4* and Liangxue Lai1,2,4*
Abstract Background: Many 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. Results: In 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. Conclusion: The 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. Keywords: Adenine and cytosine base editor (ACBE), Simultaneous C-to-T and A-to-G conversions, Mammalian systems
* Correspondence: [email protected]; [email protected] † Jingke Xie and Xingyun Huang contributed equally to this work. 1 CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated other
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