Fusion of a non-specific DNA-binding domain enhances Cas12a trans-cleavage for robust nucleic-acid diagnostics

CRISPR-Cas12a underlies powerful genome-editing and nucleic-acid detection technologies, yet its performance is limited by inefficient target engagement and low catalytic turnover, particularly at low target abundance and elevated temperatures. Here, we report a modular protein-engineering strategy to enhance Cas12a trans-activity by fusing the hyperthermophilic DNA-binding protein Sso7d to the N terminus of Lachnospiraceae bacterium ND2006 Cas12a (LbaCas12a). The resulting fusion enzyme shows a twofold improvement in detection sensitivity, a 4.6-fold increase in kcat, and substantially accelerated trans-cleavage kinetics compared with wild-type Cas12a. These enhancements are observed across multiple guide RNAs, diverse DNA substrates, and a broad temperature range from 37 to 60 {degrees}C. The engineered Cas12a enables robust detection of the intrinsic blaOXA-51 and acquired blaOXA-24 antibiotic resistance genes from multiple Acinetobacter baumannii strains with independently validated resistance profiles, whereas wild-type Cas12a produces little or no detectable signal. Signal output is increased by up to [~]30-fold, expanding the effective detection window and shortening time-to-positive. Together, these results establish fusion of an accessory DNA-binding domain as a proof-of-concept for Sso7d fusion to LbCas12a and that generalization to other CRISPR effectors or non-specific DNA-binding domains represents an exciting avenue for future investigation.