The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.

新型基因组编辑器的持续发展需要一种通用方法来分析其脱靶效应。在这里，我们描述了一种称为 Tracking-seq 的多功能方法，用于原位识别脱靶效应，该方法广泛适用于常见的基因组编辑工具，包括 Cas9、碱基编辑器和 Prime 编辑器。 Tracking-seq通过跟踪复制蛋白A（RPA）结合的单链DNA，然后构建链特异性文库，Tracking-seq需要低细胞输入，适用于体外、离体和体内基因组编辑，提供了灵敏且实用的方法。用于各种情况下脱靶检测的全基因组方法。我们证明，使用相同的引导RNA，Tracking-seq 可以检测不同编辑模式之间以及不同细胞类型之间脱靶效应的异质性，这强调了在原始系统中直接测量的必要性。