A rapid and highly sensitive CRISPR-Cas12a ortholog-assisted assay for genotyping of myostatin knockout pigs

CRISPR technology has profoundly transformed both genome editing and the next generation of nucleic acid detection. In this study, we characterized a Cas12a ortholog, Gs12-9 (EsoCas12a), which recognizes a broad PAM motif (NYYN, where Y denotes C or T) and maintains robust cis- and trans-cleavage activity across a wide temperature range (16–60 ℃), outperforming established Cas12a variants such as LbCas12a. Leveraging Gs12-9, we established a rapid, highly sensitive, and temperature-tolerant nucleic acid detection platform that enables precise genotyping without stringent thermal control. By integrating recombinase polymerase amplification (RPA) with CRISPR/Gs12-9-mediated detection, we accurately discriminated myostatin (MSTN) knockout pigs from wild-type individuals, achieving a detection limit of 40 copies per reaction with 100% accuracy. While the MSTN knockout (KO) pig served as a proof-of-concept model in this work, the versatility of Gs12-9 supports its broader application in CRISPR-based diagnostics, including pathogen screening and genetic variant detection.