CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection.

It is urgent to find an avian influenza A H7N9 detection simple method which is suitable for on-site detection. The Cas13a protein just likes a nanomachine, when specifically bound to target RNA by single-stranded RNA (crRNA), changes its protein structure and produces RNase activity, which degrades RNA non-specifically. Harnessing Cas13a, the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method. LwCas13a protein nanomachine was expressed in a prokaryotic expression system and purified by nickel column. In vitro transcribed RNA of H7N9 HA gene has been used as a target, to design a specific crRNA. The activity of Cas13a was verified with a single-stranded RNA-bound fluorescent group and a quenching fluorophore as signals. Using Cas13a, a room temperature H7N9 detection system was established. Detection of 1 nm of single-stranded RNA can be done within 5 min. When combined with the RT-RPA and T7 transcription system at room temperature, the detection limits of HA and NA are 1 fM and the reaction time is 50 min. Excellent specificity was achieved by comparison with subtype viruses such as H1N1 and H5N1. The rapid detection method based on CRISPR-Cas13a nanomachine H7N9 has been successfully established, which can detect H7N9 quickly and specifically. In the future, it can be quickly detected in the field with portable fluorescence detector.