A CRISPR-driven colorimetric code platform for highly accurate telomerase activity assay

Telomeric repeat amplification protocol (TRAP) has been the most widely used method for assessing the telomerase activity from cells and tissues. However, cell lysates, body fluid samples, or tumor tissue samples often contain high concentrations of protein or other complex matrices, which are usually inhibiting the TRAP response, thus leading to false-negative results. Internal control (IC) involved TRAP enables reliable telomerase activity assay but requires time consuming and laborious electrophoretic separation to visualize telomeric repeat DNA and internal control products from TRAP reaction, severely limiting its application in clinical diagnosis. Herein, a colorimetric code system based on programmable CRISPR-Cas12a technology and gold nano-particles (AuNPs) probe has been developed to analyse telomeric repeat DNA and internal control in TRAP products, enabling the rapid detection of telomerase activity and identification of false-negatives with naked-eye. We transform the detection results into three typical colorimetric codes-positive (P), negative (N) and false-negative (FN), making the judgement of detection results more convenient and user-friendly. The platform has also been applied in accurate detection of clinical liver cancer specimens for telomerase activity with a detection sensitivity of 93.75% and a specificity of 93.75% based on Youden index analysis. As a proof of concept, we further demonstrated the feasibility of Cas9-mediated triple-line lateral flow assay (TL-LFA), which enabled the detection of telomeric repeat DNA and internal control on a single triple-line test strip, achieving convenient and accurate telomerase activity assay.