Author:
Xie Jingke,Huang Xingyun,Wang Xia,Gou Shixue,Liang Yanhui,Chen Fangbing,Li Nan,Ouyang Zhen,Zhang Quanjun,Ge Weikai,Jin Qin,Shi Hui,Zhuang Zhenpeng,Zhao Xiaozhu,Lian Meng,Wang Jiaowei,Ye Yinghua,Quan Longquan,Wu Han,Wang Kepin,Lai Liangxue
Abstract
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.
Funder
National Key Research and Development Program of China Stem Cell and Translational Research
National Natural Science Foundation of China
Key Research & Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory
Youth Innovation Promotion Association of the Chinese Academy of Sciences
Science and Technology Planning Project of Guangdong Province
Science and Technology Program of Guangzhou
Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences
Publisher
Springer Science and Business Media LLC
Subject
Cell Biology,Developmental Biology,Plant Science,General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Physiology,Ecology, Evolution, Behavior and Systematics,Structural Biology,Biotechnology
Cited by
38 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献