UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution
UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution
复制标题
UdgX 介导的单核苷酸分辨率尿嘧啶测序。
DOI：
10.1021/jacs.1c11269
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发表时间：
2022-01-16
影响因子：
15
通讯作者：
Chen, Yi-Han
中科院分区：
文献类型：
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作者：
Jiang, Liudan;Yin, Jiayong;Chen, Yi-Han
尿嘧啶作为DNA中的一种异常碱基，可通过脱氧尿苷（dU）的错误掺入或胞嘧啶脱氨基作用产生，参与多种生理和病理过程。尿嘧啶的全基因组分布对于这些过程的研究是重要的。目前尿嘧啶全基因组定位的方法都依赖于尿嘧啶-DNA N-糖基化酶（UNG），并且在分辨率、特异性和/或灵敏度方面受到限制。在这里，我们开发了UdgX交联和聚合酶停止测序（"Ucaps-seq"）方法来以单核苷酸分辨率检测dU。首先，在合成DNA上证实了Ucaps-seq的特异性。然后在两个不同来源的基因组上验证了该方法的有效性。Ucaps-seq不仅鉴定了尿嘧啶总体升高的培美塞治疗的癌细胞中dT位点处的dU富集，而且还检测了特定区域中dU增加的活化B细胞中"WRC"基序内dC位点处的dU。最后，利用Ucaps-seq来检测由胞嘧啶碱基编辑器（nCas9-APOBEC）引入的dU，并在细胞背景下鉴定了新的脱靶位点。总之，Ucaps-seq是一个强大的工具，具有许多潜在的应用，特别是在评估碱基编辑保真度。
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.