Abstract
Fusing apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like cytidine deaminase with catalytically impaired Cas proteins (e.g., nCas9 or dCas9) provides a novel gene-editing technology, base editing, that grants targeted base substitutions with high efficiency. However, genome-wide and transcriptome-wide off-target mutations are observed in base editing, which raises safety concerns regarding therapeutic applications. Previously, we developed a new base editing system, the transformer base editor (tBE), to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations both in mammalian cells and in mice. Here we describe a detailed protocol for the design and application of the tBE. Steps for designing single-guide RNA (sgRNA) and helper sgRNA pairs, making constructs, determining the genome-wide and transcriptome-wide off-target mutations, producing the tBE-containing adeno-associated viruses, delivering adeno-associated viruses into mice and examining the in vivo editing effects are included in this protocol. High-precision base editing by the tBE can be completed within 2–3 weeks (in mammalian cells) or within 6–8 weeks (in mice), with sgRNA–helper sgRNA pairs. The whole process can be collaboratively accomplished by researchers using standard techniques from molecular biology, bioinformatics and mouse husbandry.
Key points
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This protocol describes the transformer base editor system to induce efficient editing with no observable genome-wide or transcriptome-wide off-target mutations, both in mammalian cells and in mice.
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The transformer base editor system overcomes the problems of guide RNA-independent and guide RNA-dependent off-target editing as it remains inactive at off-target sites but can be transformed to be active for base editing after binding at the on-target site.
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Data availability
The data used to generate the example results shown in Fig. 5 were originally published in ref. 27. All sequencing datasets have been deposited in the Gene Expression Omnibus under the accession code GSE164837 and GSE164477, at the NCBI BioProject under the accession code PRJNA692761 and in the National Omics Data Encyclopedia under the accession codes OEP001688, OEP001689 and OEP001690. All other data supporting the finding of this study are available from the corresponding authors on reasonable requests. Source data are provided with this paper.
Code availability
The custom Perl and Shell scripts for CFBI are available at GitHub (https://github.com/YangLab/CFBI). The computational pipeline of BEIDOU to identify high-confidence base substitution or indel events from WGS data is available at GitHub (https://github.com/YangLab/BEIDOU). The workflow of RADAR to detect and visualize all 12 possible types of RNA-editing event from RNA-seq data is available at GitHub (https://github.com/YangLab/RADAR).
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Acknowledgements
This work was supported by 2018YFA0801401 (J.C.) and 2019YFA0802804 (L.Y.) from the National Key R&D Program of China, 81872305 (J.C.) and 31925011 (L.Y.) from the National Natural Science Foundation of China, NK2022010207 (J.C.) from Ministry of Agriculture and Rural Affairs, 21JC1404600 (J.C.), 20PJ1410200 (J.L.) and 23XD1422500 (J.C.) from Shanghai Municipal Science and Technology Commission. We thank Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine, ShanghaiTech University, Shanghai Clinical Research and Trial Center, Animal Core Facility, ShanghaiTech University and Molecular and Cell Biology Core Facility, School of Life Science and Technology, ShanghaiTech University for providing support.
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J.C., L.Y. and J.L. conceived, designed and supervised the project. W.H., B.-Q.G., J.Z., Z.H., J.L., L.Y. and J.C. wrote the paper.
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Nature Protocols thanks Yongsub Kim, Liangxue Lai, Zhanjun Li, Shaohua Yao, Erwei Zuo and the other, anonymous, reviwer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Wang, L. et al. Nat. Cell Biol. 23, 552-563 (2021): https://doi.org/10.1038/s41556-021-00671-4
Extended data
Extended Data Fig. 1 Procedure to construct the designed plasmids each of which expresses a pair of hsgRNA-MS2 and sgRNA-boxB.
a, The schematic diagram demonstrating the processes of constructing one plasmid expressing a pair of designed hsgRNA-MS2 and sgRNA-boxB. b, The schematic diagram demonstrating the construction of plasmids designed for the screening of all combinations of sgRNAs and hsgRNAs against one on-target site.
Supplementary information
Supplementary Table 1
Supplementary Table 1.
Source data
Source Data Fig. 5
Statistical source data.
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Han, W., Gao, BQ., Zhu, J. et al. Design and application of the transformer base editor in mammalian cells and mice. Nat Protoc 18, 3194–3228 (2023). https://doi.org/10.1038/s41596-023-00877-w
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DOI: https://doi.org/10.1038/s41596-023-00877-w
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