Enhanced One‐Pot Cas12a‐Based Nucleic Acid Detection via Epitope Insertion and Recruitment of Rad51

The CRISPR-Cas12a system has emerged as a promising tool for nucleic acid-based diagnostics. However, its multi-step workflow and limited sensitivity hinder its integration into point-of-care testing (POCT). Here, the ECOT system (Engineered Cas12a for One-pot Test), a novel approach that combines protein engineering with one-pot detection, offering high sensitivity, specificity, and rapid response is introduced. By introducing GCN4 epitope insertions into LtCas12a and LbCas12a variants, their cis-cleavage activity, promoting efficient accumulation of amplification products is reduced. Additionally, the inclusion of scFv-Rad51 (single-chain variable fragment-Rad51) enhances Cas12a's trans-cleavage activity, amplifying signal intensity. The ECOT-Lb system demonstrated superior sensitivity in detecting low-copy HPV DNA samples, outperforming traditional qPCR in clinical tests. Achieving detection limits as low as 3 copies in under 30 min, the ECOT-Lb system is well-suited for home-based self-testing and widespread clinical diagnostics. This work provides a versatile and scalable protein engineering strategy that enhances the performance of CRISPR-based diagnostic tools, offering a promising platform for rapid molecular detection in diverse applications.