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UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution

As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping...

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Published in:Journal of the American Chemical Society 2022-01, Vol.144 (3), p.1323-1331
Main Authors: Jiang, Liudan, Yin, Jiayong, Qian, Maoxiang, Rong, Shaoqin, Zhang, Shengqi, Chen, Kejing, Zhao, Chengchen, Tan, Yuanqing, Guo, Jiayin, Chen, Hao, Gao, Siyun, Liu, Tingting, Liu, Yi, Shen, Bin, Yang, Jian, Zhang, Yong, Meng, Fei-Long, Hu, Jinchuan, Ma, Honghui, Chen, Yi-Han
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Language:English
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Summary:As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution, specificity, and/or sensitivity. Here, we developed a UdgX cross-linking and polymerase stalling sequencing (“Ucaps-seq”) method to detect dU at single-nucleotide resolution. First, the specificity of Ucaps-seq was confirmed on synthetic DNA. Then the effectiveness of the approach was verified on two genomes from different sources. Ucaps-seq not only identified the enrichment of dU at dT sites in pemetrexed-treated cancer cells with globally elevated uracil but also detected dU at dC sites within the “WRC” motif in activated B cells which have increased dU in specific regions. Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine base editor (nCas9-APOBEC) and identified a novel off-target site in cellular context. In conclusion, Ucaps-seq is a powerful tool with many potential applications, especially in evaluation of base editing fidelity.
ISSN:0002-7863
1520-5126
1520-5126
DOI:10.1021/jacs.1c11269