CRISPR-Cas12a coupled with terminal deoxynucleotidyl transferase mediated isothermal amplification for sensitive detection of polynucleotide kinase activity

Polynucleotide kinase (PNK) is an important DNA damage repair-related enzyme and also a promising therapeutic target in various diseases. Here, we developed a dual amplified sensing strategy based on the combination of terminal deoxynucleotidyl transferase (TdT) and CRISPR-Cas12a for high selective and sensitive detection of PNK activity in cell lysates and also inhibitor screening. In this sensing system, the PNK converted the 3'-phosphate DNA to 3'-hydroxy DNA, and then the TdT catalyzed to add a long poly-adenine (poly (A)) at the 3' OH terminus DNA (as first amplified step). The produced poly (A) tail acted as activator to trigger the Cas12a trans-cleavage (non-target) activity to cleave reporter probes for fluorescence detection (as second amplified step). The dual amplified steps lead to a synergetic signal amplification effect for sensitive detection of PNK activity. The developed sensing platform enable detect PNK activity with linear range from 0 to 1 U/mL and a direct detection limit of 5 x 10(-4) U/mL. It was also successfully used for PNK inhibitor screening and PNK activity detection in cell extracts even at a single-cell level. The efficient dual amplified method can broaden the CRISPR/Cas-based sensing systems in other bio-/chem-analysis fields.