小柯机器人

北京大学伊成器团队开发出胞嘧啶碱基编辑器全基因组脱靶效应检测的新工具。相关论文于2021年6月7日发表于国际学术期刊《自然—方法学》。

研究人员报告了用于评估胞嘧啶碱基编辑器（CBE）特异性的Detect-seq。它能够在全基因组水平上灵敏检测CBE诱导的脱靶位点。Detect-seq利用化学标记和生物素下拉来追踪编辑中间体脱氧尿苷，从而揭示CBE的编辑组。除了不依赖于Cas9和典型依赖于Cas9的脱靶位点之外，研究人员还发现了原始间隔区序列之外和目标链（与单向导RNA配对）上的编辑。这种意外的脱靶编辑很普遍，并且可以表现出很高的编辑率，而它们的发生表现出细胞类型依赖性，无法根据sgRNA序列进行预测。

此外，研究人员在测试的目标位点附近发现了原始间隔区外和目标链的编辑，这挑战了CBE不会诱导近端脱靶突变的常识。总的来说，这个方法能够对CBE编辑组进行无偏倚分析，并为各种新兴基因组编辑工具的特异性评估提供广泛适用的工具。

据了解，CBE具有纠正人类致病点突变的潜力。然而，它们的全基因组特异性仍然知之甚少。

附：英文原文

Title: Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors

Author: Zhixin Lei, Haowei Meng, Zhicong Lv, Menghao Liu, Huanan Zhao, Hao Wu, Xiaoxue Zhang, Lulu Liu, Yuan Zhuang, Kailin Yin, Yongchang Yan, Chengqi Yi

Issue&Volume: 2021-06-07

Abstract: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools. 

DOI: 10.1038/s41592-021-01172-w

Source: https://www.nature.com/articles/s41592-021-01172-w

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex