Fig. 1: Design and working principle of the AIOD-CRISPR assay.

a Schematic of the AIOD-CRISPR assay system. SSB is single-stranded DNA binding protein. The four sites in the target sequence are labeled as F, T1, T2, and R, respectively. The letter c represents the corresponding complementary site. For example, F and Fc sites are complementary. The short horizontal lines with the same colors denote the same sites and their arrows represent the direction of 5′–3′. b Evaluation of eight AIOD-CRISPR reactions (R) with various components through endpoint imaging after 40-min incubation, denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the single-stranded fluorescent reporter (ssDNA-FQ), and real-time fluorescence detection. The ssDNA-FQ was labeled by 5′ 6-FAM (Fluorescein) fluorophore and 3′ Iowa Black FQ quencher. Recombinase polymerase amplification (RPA) mix from TwistAmp Liquid Basic kit was composed of 1× Reaction Buffer, 1× Basic E-mix, 1× Core Reaction Buffer, 14 mM MgOAc, 320 nM each of primers, and 1.2 mM dNTPs. Dual crRNAs contained 0.64 μM each of crRNAs specific to the SARS-CoV-2 N gene sequence. A plasmid containing the SARS-CoV-2 N gene sequence (3 × 10³ copies), 8 μM of ssDNA-FQ reporters, and 1.28 μM EnGen Lba Cas12a (Cpf1) were used. Each experiment was repeated three times with similar results.