Dual Mechanisms of crRNA 3′-Extension-Mediated Cas12a Attenuation Enable Programmable One-Pot CRISPR Diagnostics

Precise control of Cas12a trans-cleavage activity is essential for developing robust one-pot CRISPR diagnostics, yet existing strategies remain largely empirical. In this study, we show that minimal 3′-RNA extensions of 7–13 nucleotides reliably attenuate Cas12a activity through a dual mechanism: competitive 3′extension cleavage and steric blockade of the RuvC catalytic pocket. Leveraging this mechanistic insight, we developed SMART-POT (Simple Modulation via Attenuating RNA Tail in One-POT detection), an integrated one-pot RPA-Cas12a platform that bypasses noncanonical PAM crRNA screening. Using a canonical PAM HPV18-targeting crRNA with a 7-nt extension, the assay achieved 10 –17 M sensitivity, perfect specificity against 13 other high-risk HPV genotypes, and 100% clinical concordance (20/20 samples) with qPCR. This work establishes a rational design rule for Cas12a regulation and provides a generalizable framework for field-ready one-pot CRISPR diagnostics.