• CRATS integrates CRISPR-Cas12b and RPA in a one-pot reaction using a temperature switch (37°C for amplification, 60°C for cleavage), overcoming the challenge of interference between Cas activity and amplification.
• The assay achieves high sensitivity with a detection limit of 100 copies of monkeypox virus DNA per reaction, and demonstrates good specificity.
• The temperature-switch strategy provides a novel and convenient approach for one-pot CRISPR-based diagnostics, eliminating the need for separate steps and reducing cross-contamination risk.
• CRATS offers a point-of-care testing (POCT) friendly tool for rapid and sensitive detection of monkeypox virus, addressing urgent public health needs.