UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Uracil is an aberrant DNA base arising from deoxyuridine misincorporation or cytosine deamination.
• Genome-wide uracil profiling is crucial for understanding its role in physiological and pathological processes.
• Existing methods for mapping uracil have limitations in resolution, specificity, and sensitivity.

Purpose of the Study:

• To develop a novel, high-resolution method for detecting uracil in whole genomes.
• To overcome the limitations of current uracil detection techniques.

Main Methods:

• Development of UdgX cross-linking and polymerase stalling sequencing (Ucaps-seq).
• Validation of Ucaps-seq specificity using synthetic DNA.
• Application of Ucaps-seq to diverse genomic samples, including cancer cells and activated B cells.
• Utilizing Ucaps-seq to assess the fidelity of cytosine base editors.

Main Results:

• Ucaps-seq demonstrated single-nucleotide resolution for uracil detection.
• The method confirmed enrichment of uracil at deoxythymidine sites in cancer cells treated with pemetrexed.
• Ucaps-seq identified uracil at deoxycytidine sites within WRC motifs in activated B cells.
• A novel off-target site for nCas9-APOBEC cytosine base editor was identified in a cellular context.

Conclusions:

• Ucaps-seq is a powerful and versatile tool for genome-wide uracil mapping.
• The method offers significant improvements in resolution, specificity, and sensitivity compared to existing techniques.
• Ucaps-seq has broad applications, particularly in evaluating the fidelity of base editing technologies.