Due to an extreme rarity of 5-carboxylcytosine (5caC) in the mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental and computational resources, increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high-resolution mapping of 5caCGs. The method uses an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC, generating unmodified CGs, which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supports the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflect their distinct demethylation dynamics and demonstrate an association of 5caC with gene expression. Generally, we demonstrate that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC.

由于5-羧基胞嘧啶（5caC）在哺乳动物基因组中极为稀有，对其作用的研究带来了相当大的挑战。已经提出了基于亚硫酸氢盐测序的方法用于全基因组5caC分析。但是，基于亚硫酸氢盐的几乎不丰富的5caC测序需要大量的实验和计算资源，从而增加了测序成本。在这里，我们提出了一种无亚硫酸氢盐的方法caCLEAR，用于5caCG的高分辨率映射。该方法利用甲基转移酶eM.SssI的非典型活性从5caC中除去羧基，生成未修饰的CG，这些CG通过uTOP-seq测序定位。在模型DNA系统和小鼠ESC上对caCLEAR的验证支持了caCLEAR对5caCGs分析的适用性。天真和引发的多能ESC的5caCG谱反映了它们独特的去甲基化动力学，并证明了5caC与基因表达的关联。一般而言，我们证明caCLEAR是一种可靠的经济方法，可以帮助您深入了解5caC的生物学作用。