清华大学蓝勋、李寅青研究组合作的最新研究利用Tracking-seq揭示出CRISPR-Cas9介导基因组编辑中脱靶效应的异质性。相关论文于2024年7月2日发表在《自然-生物技术》杂志上。

研究人员开发了一种名为跟踪-质谱（Tracking-seq）的多功能方法，用于原位鉴定脱靶效应，这种方法广泛适用于常见的基因组编辑工具，包括Cas9、碱基编辑器和质粒编辑器。通过追踪复制蛋白A（RPA）结合的单链DNA，然后构建链特异性文库，Tracking-seq只需很少的细胞，适用于体外、活体外和体内基因组编辑，为不同情况下全基因组的脱靶检测提供了一种灵敏、实用的方法。

研究表明，使用相同的引导RNA，Tracking-seq能检测到不同编辑方式和不同细胞类型之间的脱靶效应异质性，从而证明了在原始系统中进行直接测量的必要性。

研究人员表示，新型基因组编辑器的发展需要一种通用方法来分析它们的脱靶效应。

附：英文原文

Title: Tracking-seq reveals the heterogeneity of off-target effects in CRISPR–Cas9-mediated genome editing

Author: Zhu, Ming, Xu, Runda, Yuan, Junsong, Wang, Jiacheng, Ren, Xiaoyu, Cong, Tingting, You, Yaxian, Ju, Anji, Xu, Longchen, Wang, Huimin, Zheng, Peiyuan, Tao, Huiying, Lin, Chunhua, Yu, Honghao, Du, Juanjuan, Lin, Xin, Xie, Wei, Li, Yinqing, Lan, Xun

Issue&Volume: 2024-07-02

Abstract: 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.

DOI: 10.1038/s41587-024-02307-y

Source: https://www.nature.com/articles/s41587-024-02307-y