Chemical Modification Coupled with Isothermal CRISPR-Based Assay for Sensitive Detection of DNA Hydroxymethylation

5-Hydroxymethylcytosine (5hmC) plays a key role in the DNA demethylation process and serves as a stable epigenetic marker in the human genome which is closely associated with disease progression, particularly in diabetes, colorectal cancer, and liver cancer. However, convenient and sensitive methods for detecting and quantifying 5hmC in the genome are scarce, especially in complex biological environments. Herein, a novel attempt at hypersensitive quantitative detection of 5hmC was presented. A multifunctional photosensitive probe was therefore introduced for specific labeling, enrichment, and elution of 5hmC-DNA. Combining with isothermal assay leveraging rolling circle amplification and Cas12a for accurate recognition, we achieved quantitative detection of 5hmC DNA in trace amounts at a level of 11 fM. Global 5hmC was measured in various biological samples using as little as 10 ng of input DNA by a real-time PCR instrument. The reported approach imposed no sequence restrictions, demonstrating promising potential for detecting modified bases in trace amounts of nucleic acids within complex environments, such as blood, urine, and saliva samples.