OPTIMIZATION OF CRISPR/CAS12A TRANS-CLEAVAGE REACTION FOR DETECTION OF THE STAPHYLOCOCCUS AUREUS NUCA GENE

Staphylococcus aureus (S. aureus) is the most prevalent pathogen within the staphylococcal group, classified as a gram-positive bacterium responsible for severe infections affecting the skin, bloodstream, organs, and gastrointestinal tract. Consequently, the development of early and accurate detection tools is a research priority. Recently, CRISPR/Cas technology, particularly Cas12a, has emerged as a promising diagnostic tool due to its ability to trans-cleave single-stranded DNA (ssDNA) following the specific cleavage of double-stranded DNA (dsDNA) targets. In this study, we optimized a CRISPR/Cas12a trans-cleavage reaction for detecting the nucA gene, a specific marker of S. aureus. To enhance enzyme activity, four crRNAs (crRNA1–4) were designed and tested, each exhibiting different effects on the reaction. However, a mixture of three crRNAs demonstrated higher efficiency than reactions using a single crRNA. Additionally, key reaction parameters, including enzyme concentration, additives, probe sequence, and incubation temperature, were systematically evaluated. The optimized reaction conditions included 6.25 nM LbCas12a combined with the mixture of three crRNAs (crRNA1–3) in a reaction containing 10 mM dithiothreitol (DTT) and 1 µM TA5C ssDNA FAM-BHQ1 probe. The assay was incubated at 39°C for 30 minutes, achieving a detection limit of 284.8 amol or 1.72 × 10⁸ copies of the nucA gene. Fluorescent signals were detectable under a standard UV transilluminator. This study provides valuable insights for the development of CRISPR-based diagnostic kits for S. aureus detection.