Indiscriminate Trans-Cleavage Activity of CRISPR/SuCas12a2 Enables Sensitive Detection of SARS-CoV-2.

Sensitive detection of SARS‑CoV‑2 remains critical for controlling COVID‑19 outbreaks and guiding patient care. Although reverse transcription-polymerase chain reaction (RT‑PCR), the gold standard for detecting SARS-CoV-2, is highly sensitive, the need for specialized equipment and trained personnel limits its widespread application in low or middle-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology could overcome these limitations by providing simplicity, low cost, and high specificity. However, current CRISPR-based diagnostics can simultaneously cleave the target and fluorescence probes, as they are the same nucleic acid type (ssDNA or ssRNA), thereby reducing detection sensitivity. Herein, we developed a novel CRISPR-based viral detection method using SuCas12a2 (Cas12a2 from Sulfuricurvum sp. PC08-66), which harnesses its unique broad trans-cleavage activity and offers flexibility in selecting fluorescence probes. Using the conserved SARS‑CoV‑2 envelope gene as the model analyte, the analytical performance of the CRISPR/SuCas12a2 system for viral detection was evaluated. The CRISPR/SuCas12a2 detection workflow achieved a detection limit of 5 × 103 copies/μL for SARS-CoV-2 viral RNA. When detecting nasopharyngeal swab samples from patients, the CRISPR/SuCas12a2 system showed preliminary agreement with RT-qPCR in a set of clinical samples. Our CRISPR/SuCas12a2 system provides a flexible detection platform with simplified probe selection and enhanced compatibility, offering new insights into future portable diagnostic applications and enhancing global public health surveillance.