The CRISPR/Cas12a system has demonstrated extraordinary capabilities in biosensing and molecular diagnostics, owing to its precise recognition and efficient trans-cleavage ability. However, current crRNA-based Cas12a regulation is relatively crude, requiring costly modifications and multiple components, increasing system complexity. Here, we develop translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a activity (THRUST), a powerful and economical Cas12a regulation strategy. This strategy utilizes a bifunctional template-activator construct (TAC) that simultaneously functions as a transcriptional template for T7 RNA polymerase (T7 RNAP) and an activator for Cas12a. T7 RNAP skips the deoxyuridine (dU) lesion while being blocked by the apurinic/apyrimidinic (AP) site. Strategic positioning of transcriptional regulatory units on the TAC allows precise control of crRNA length and simultaneously regulates Cas12a activation at the activator level, thereby achieving hierarchical regulation of Cas12a. Through the construction of “Dim down” and “Light up” biosensing platforms and an aggregation-induced emission lateral flow test, THRUST enriches the CRISPR/Cas12a regulatory toolbox for molecular diagnostics.
