Digital CRISPR/Cas12a-based platform for precise quantification of telomerase activity

Precise quantification of telomerase activity is of great significance for telomerase-based early cancer diagnosis, as well as the screening of prodrugs. Telomeric repeat amplification protocol (TRAP) has been most widely used for assessing telomerase activity, which is limited by false-positive results, complicated operations, and long detection time. Herein, we develop a droplet digital hyperbranched amplification enhanced CRISPR/Cas12abased telomerase detection platform (ddHECTelo) for precise quantitative detection of telomerase activity. By combining with the droplet digital platform and hyperbranched amplification, the ddHECTelo allows precise quantification of telomerase activity with single-molecule sensitivity. A polydisperse droplets digital system was employed, so that the reaction mixtures could be partitioned into picoliter-scale polydisperse droplets in several seconds to avoid the pre-amplification, without the need for a sophisticated microfluidic chip. This is the first digital isothermal platform for sensitive and precise quantification of telomerase activity, which shows great potential in telomerase-based early cancer diagnosis. It could also be used to analyze the telomerase activity in fundamental biomedical research.