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.

碱基编辑器可以实现有针对性的基因组碱基转换。然而，脱靶问题是其应用中的主要问题之一。个体水平的全基因组测序 (WGS) 可以提供有关全基因组特异性的直接信息，但很难将碱基编辑器诱导的真正脱靶单核苷酸变异 (SNV) 与背景变异区分开来。在这里，我们描述了一种用于评估水稻碱基编辑器特异性的无偏 WGS 方法。在该协议中，我们描述了实验设计并提供了载体构建、水稻转化和组织培养的详细信息，以及用于克服各种植物物种中两个相关核心问题的综合 WGS 数据分析管道：高背景突变率和异质性检查的人群。使用该协议，