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Probing low abundant DNA methylation by CRISPR-Cas12a-assisted cascade exponential amplification.

Liangliang ZhangXianxian ZhaoXiaolin HuYi ZhangRuining LiuHai PengYouhao ChenHong ZhangYang Luo
Published in: The Analyst (2022)
Aberrant DNA methylation plays a pivotal role in tumor development and metastasis, and is regarded as a valuable non-invasive cancer biomarker. However, the sensitive and accurate quantification of DNA methylation from clinical samples remains a challenge. Herein, we propose an easy-to-operate Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system Assisted Methylation (CAM) approach for the sensitive detection of DNA methylation through the integration of rolling circle amplification and CRISPR-Cas12a-assisted cascade amplification. Briefly, bisulfite was employed to prepare the clinical samples so that the methylated DNA sequences trigger the subsequent triple signal amplifications, whilst the normal counterparts do not. The triple signal amplification procedure consists of methylated DNA sequence-based rolling circle amplification for a preliminary signal enhancement, a nicking enzyme-initiated target cleavage for a secondary amplification, and CRISPR-Cas12a enzyme-mediated trans-cleavage for a tertiary signal enhancement. This proposed approach reveals high sensitivity, which can even distinguish as low as 0.01% methylation levels from mixtures, paving the way towards the acceleration of methylation-based cancer diagnostics and management.
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