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 (LbCas12a). The resulting fusion enzyme shows a twofold improvement in detection sensitivity, a 4.6-fold increase in k cat, app , and a fivefold reduction in the time to signal plateau (3 versus 15 min) compared with wild-type Cas12a. These enhancements are guide RNA-dependent (two of the four guide RNAs tested), are retained across diverse DNA substrates and within a working temperature range of 37 to 60 °C, above which activity is lost. The engineered Cas12a enables robust detection of the intrinsic bla OXA−51 and acquired bla OXA−24 antibiotic resistance genes from multiple Acinetobacter baumannii strains with independently validated resistance profiles, whereas wild-type Cas12a produces little or no detectable signal, raising the signal-to-noise ratio by up to ~ 30-fold. Together, these results identify N-terminal fusion via an XTEN linker as the productive architecture for enhancing Cas12a trans-cleavage and define the conditions under which an accessory DNA-binding domain improves a CRISPR diagnostic effector.