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

Abstract 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.