Sensitive and amplification-free detection of telomerase activity by self-extension of telomerase and trans-cleavage of CRISPR/Cas12a

Abstract

Telomerase is a well-known cancer biomarker, and the reliable and sensitive detection of telomerase activity is of vital significance for cancer diagnosis and treatment. Despite many attempts, the sensitive methods for telomerase activity assay mainly depend on the various nucleic acid amplification, which requires complex primers/probes design and multiple enzymes. In addition, the multiple experimental procedures may introduce false-positive results from cross-contamination of amplification products challenging the reliability of telomerase-based diagnostics detection. Herein, we present a reliable, amplification-free, and sensitive strategy for the detection of telomerase activity based on the self-extension of telomerase and trans-cleavage of CRISPR/Cas12a. In this work, crRNA/Cas12a complexes can specifically target the self-extending telomere-repetitive DNA and activate the typical CRISPR/Cas12a-based fluorescent output system, which allows the detection of telomerase activity in very few cancer cells (5 HeLa cells) without complex primer design and any nucleic acid amplification. Furthermore, the proposed method holds high reliability and specificity in distinguishing tumor cells from normal cells. With these unique features, including high sensitivity and reliability, excellent specificity, and simplicity, this method highlights a great potential for telomerase-related research and broadens the CRISPR-Cas12a-based sensing toolbox.