2. Academic Validation
  3. An unbiased method for evaluating the genome-wide specificity of base editors in rice

An unbiased method for evaluating the genome-wide specificity of base editors in rice

  • Nat Protoc. 2021 Jan;16(1):431-457. doi: 10.1038/s41596-020-00423-y.
Shuai Jin 1 2,  Qiang Gao 3,  Caixia Gao 4 5
Affiliations

Affiliations

  • 1 State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
  • 2 College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
  • 3 BGI Genomics, BGI-Shenzhen, Shenzhen, China.
  • 4 State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.
  • 5 College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. cxgao@genetics.ac.cn.
PMID: 33349703 DOI: 10.1038/s41596-020-00423-y
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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