CRISPR-Cas-Based Pen-Side Diagnostic Tests for and .

and are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of and in infected animals relies primarily on microscopic examination of Giemsa-stained blood or organ smears, which has limited sensitivity. Molecular methods offer higher sensitivity but are costly and impractical in resource-limited settings. Following the development of a pen-side test for detecting infections in cattle, we have created two additional CRISPR-Cas12a assays targeting and . The assays target the major surface protein 5 (MSP5) for and rhoptry-associated protein 1a (RAP1a) for . These additional tests involve a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a detection with a lateral strip readout. Results demonstrate high specificity, with no cross-reactivity against other tick-borne parasites, and a limit of detection down to 10 DNA copies/µL of each target marker. The findings pave the way for sensitive and user-friendly pen-side tests to diagnose and infections.