A multiplexed TSA/CRISPR-mediated one-pot system for rapid d

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crRNA2 at25, 30, and 15 min, respectively, exhibiting higher fluorescence intensity than others (Fig. 1C). In summary, the optimal crRNAs identified included SARS-CoV-2 crRNA3, RV crRNA1, MPXV F3L crRNA1 and MPXV B6R crRNA2.A series of optimizations were implemented to enhance the detection sensitivity of TCOPS. The efficacy of the one-pot system is determined by two vital factors, the concentration of the RNP solution and fluorescent reporter genes (Lin et al., 2023). Serving as a cleavage template for the Cas12/crRNA complex, RNP is dynamically balanced with TSA, facilitating a rapid one-pot reaction (Li et al., 2024). In the present study, we tested fluorescence signals with RNP concentrations of 12.5, 25, 50, and 100 nM. The highest signal was detected at 100 nM, which was then adopted in TCOPS (Fig. 2A, D). The length and composition of reporter genes critically influence Cas12a's trans-cleavage activity; shorter reporters (e.g., 5 nt) may improve reaction kinetics but reduce signal amplitude, while longer reporters (e.g., 8 nt) typically offer stronger fluorescence accumulation and stability (Lee et al., 2022). To determine the optimal design for our one-pot system, we systematically compared 5-nt (5’-FAM-TTATT-BHQ1–3′) and 8-nt (5’-FAM-TTATTATT-BHQ1–3′) reporters across a final range of concentrations (200–800 nM). Our data confirmed that the 8-nt reporter provided significantly stronger signals at 800 nM (Fig. 2B, C, E). The highest fluorescence signal was achieved using 100 nM RNP and a final concentration of 800 nM 8-nt ssDNA reporter, which were subsequently adopted in TCOPS.