A split single-stranded DNA activator-based Cas12a fluorescence biosensor for specific H1N1 detection

We developed a novel CRISPR/Cas12a fluorescence biosensor to specifically detect the H1N1 virus based on splitting single-stranded DNA (ssDNA) activators. Specifically, activators 1 and 2 were generated from dual target nucleic acid sites to induce a three-way junction (3WJ) isothermal amplification process. Two specific H1N1 RNA nucleic acid sites were bound to a set of probes to form a 3WJ, which facilitated repeated extension and nicking reactions to produce high levels of split ssDNA activators via DNA polymerase and nicking endonuclease activity. Amplified activators 1 and 2 fully assembled with Cas12a/crRNA to form complexes. Activated Cas12a transcleaved ssDNA reporter molecules to generate fluorescence signals. Under optimal conditions, biosensor dynamic detection range was 1 pM-10 nM, and the detection limit was 205 fM. Thus, our biosensor showed high specificity, reliability, and robustness, which highlights its potential use for H1N1 diagnostics.