Fig. 6: Assessment of the CRISPR-drCas12f1/Cas13a system for detection of the human POP7 gene and IVA/H1N1 virus.

a Schematic of the dual-gene detection method utilizing the CRISPR-drCas12f1/Cas13a system. Isothermal amplification of two genes is achieved through multiplexed RT-RPA, employing two specific primer sets. One primer in each set is modified with PT to protect the target strand from T7 exonuclease degradation. Specific gRNAs target these PT-modified ssDNA, initiating drCas12f1 and Cas13a’s trans-cleavage activities, respectively cleaving ssDNA reporters (VIC-TTTTTTTTTTTT-BHQ1) and RNA reporters (FAM-rUrUrUrUrUrUrUrUrU-BHQ1), resulting in the emission of fluorescence signals at distinct wavelengths. Created in BioRender (Wu, Q. (2024) BioRender.com/l69i026). b Demonstrates the four potential outcomes of the CRISPR-drCas12f1/Cas13a-based dual-gene detection strategy. The final concentration of the ssDNA targets was: POP7 (5 nM), and H1 (1 nM). “VIC” and “FAM” indicate different fluorescence channels. Raw fluorescence intensities over 10 min are shown. The connecting lines represent mean values, and each circle represents an individual data point. c Determination of the detection limit for the CRISPR-drCas12f1/Cas13a system targeting human POP7 RNA and IVA/H1N1 standard RNA. d Specificity assessment of the CRISPR-drCas12f1/Cas13a system for detecting human POP7 RNA and IVA/H1N1 standard RNA against other influenza viruses. The final concentration of human POP7 RNA was 10 nM, and all influenza viruses were at a concentration of 10,000 copies per μL. “VIC” and “FAM” indicate different fluorescence channels. e, f Utilization of the CRISPR-drCas12f1/Cas13a system for detection of the human POP7 gene (e) and the H1 gene of IVA/H1N1 virus (f) in 30 throat swab clinical samples. A blue threshold line indicates the mean of fluorescence intensity from a non-target control (NTC). RT-qPCR detected 25 clinical IVA/H1N1 positive samples and 5 negative samples, with “Sample ID” and “Ct” values provided. Purple asterisks mark samples verified as IVA/H1N1 negative by RT-qPCR. Un, Undetermined. b–f Raw fluorescence intensities at 10 min are presented. NTC, no target control. a.u., arbitrary units. n = 3 biologically independent experiments. Error bars represent mean ± SEM. Statistical significance was determined using unpaired two-tailed Student’s t-tests: ns (not significant, p > 0.05); **(p < 0.01); ***(p < 0.001); ****(p < 0.0001).