基因组编辑
计算生物学
胞嘧啶
生物
清脆的
点突变
序列(生物学)
DNA
基因组
Cas9
遗传学
突变
基因
作者
Zhi‐Xin Lei,Haowei Meng,Zhicong Lv,Menghao Liu,Huanan Zhao,Hao Wu,Xiaoxue Zhang,Lulu Liu,Yuan Zhuang,Kang Yin,Yongchang Yan,Chengqi Yi
出处
期刊:Nature Methods
[Springer Nature]
日期:2021-06-01
卷期号:18 (6): 643-651
被引量:36
标识
DOI:10.1038/s41592-021-01172-w
摘要
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. Detect-seq, built upon chemical labeling and enrichment of intermediate deoxyuridine, offers an approach to profile different types of off-target mutation induced by cytosine base editors including unexpected edits outside of protospacer and on the target strand.
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