Abstract
Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12–15 weeks.
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Data availability
All the sequence data have been deposited in NCBI BioProject under accession code PRJNA522656, in which CBE refers to BE3 and HF1-CBE to HF1-BE3.
Code availability
All the code used in this protocol is available on GitHub at: https://github.com/ReiGao/GWSBE. The code in this protocol has been peer-reviewed.
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Acknowledgements
We thank Y. Zhang (Department of Plant and Environmental Sciences, University of Copenhagen) for critical revision of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31788103), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102), the National Key Research and Development Program of China (2016YFD0101804) and the R&D Program in Key Areas of Guangdong Province (2018B020202005).
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S.J. performed the experiments; S.J. designed figures; C.G. supervised the project; S.J., Q.G. and C.G. wrote the manuscript.
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Peer review information Nature Protocols thanks Keiji Nishida, Pengcheng Wei and Huanbin Zhou for their contribution to the peer review of this work.
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Key reference using this protocol
Jin, S. et al. Science 364, 292–295 (2019): https://doi.org/10.1126/science.aaw7166
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Supplementary Figs. 1 and 2, Supplementary Notes 1–3 and Supplementary Tables 1–4.
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Jin, S., Gao, Q. & Gao, C. An unbiased method for evaluating the genome-wide specificity of base editors in rice. Nat Protoc 16, 431–457 (2021). https://doi.org/10.1038/s41596-020-00423-y
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DOI: https://doi.org/10.1038/s41596-020-00423-y
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