UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution
| Autor: | Liudan Jiang, Jiayong Yin, Maoxiang Qian, Shaoqin Rong, Shengqi Zhang, Kejing Chen, Chengchen Zhao, Yuanqing Tan, Jiayin Guo, Hao Chen, Siyun Gao, Tingting Liu, Yi Liu, Bin Shen, Jian Yang, Yong Zhang, Fei-Long Meng, Jinchuan Hu, Honghui Ma, Yi-Han Chen |
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| Rok vydání: | 2022 |
| Předmět: | |
| Zdroj: | Journal of the American Chemical Society. 144:1323-1331 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/jacs.1c11269 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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