Extraction of PCO13-Twinstrep-SUMO-huLwCas13a-plasmid used the Plasmid Mini Kit (OMEGA). 50ng of plasmids were then transferred into E. coli competent cells BL21 (Cwbiotech). After transformation, 100 μL of dilutions of cells were plated on ampicillin (Amp) and Luria-Bertani (LB)-agar plate and incubated overnight at 37°C. The next day, a single colony was picked and plated in a 10 ml of LB medium (Containing 100 μL of Amp), which was used to inoculate 1 L of LB medium and shook overnight at speed of 220 r/min until the OD600 was 0.6 at 37°C. At this time, LwCas13a protein expression was induced by supplementation with IPTG to a final concentration of 500 μM. The culture was maintained at 16°C for 16 h, and the cells were then centrifuged at 4,500 g for 10 min at 4°C. Considering the stability of the Cas13a protein, all steps of protein purification were carried out at 4°C. After centrifugation, the cell pellet was harvested and resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM DTT, pH 7.4). Then, protease inhibitor (MCE) and lysozyme were added, followed by sonication (JX-650, Shanghai, China) with following conditions: 200 W of power, ultrasonic 2.5 s and 10 s of pause for 30 min. Centrifugation was carried at 10,000 g for 60 min at 4°C and filtered the supernatant through a 220 nm filter (Sterile Millex). The filtered supernatant was applied to a 5 ml HisTrapHP nickel ion affinity column (GE Healthcare Life Sciences) via Fast protein liquid chromatography (FPLC) (AKTA PURE, GE Healthcare Life Sciences). Then the protein bound column was washed three times using a buffer (20 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH 7.4). About 250 Units of SUMO protease (ThermoFisher) was then loaded onto the column and incubated overnight at 4°C with rotation. Finally, the column was washed with lysis buffer again and the collected solution was analyzed by 12% SDS-PAGE. Fractions containing pure LwCas13a were pooled and exchanged the buffer via a Millipore centrifugal ultrafiltration tube (Amicon Ultra) to a storage buffer (600 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5% glycerol, 2 mM DTT). The final product was stored at −80°C.

Plasmid containing the HA or NA gene of the H7N9 virus was amplified using PrimeSTAR Max DNA Polymerase (TaKaRa), and then purified using a SanPrep PCR product purification kit (Sangon Biotech). Purified dsDNA was incubated with T7 polymerase overnight at 37°C using HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs). The RNA was purified by RNA rapid concentration and purification kit (Sangon Biotech) and quantified by One drop (American Thermal Power Corporation) to prepare a gradient RNA standard solution. The crRNA was designed based on the target RNA sequence. Two complementary crRNA-DNA primers (with an appended T7 promoter sequence) were annealed with final concentration 10 μM. Then the dsDNAs were transcribed to crRNA, using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs).

Taking 10 mg/ml carboxylated MBs was stored in a refrigerator at 4°C, followed by vortex on a vortex shaker for 5 min to mix the MBs. 100 μL of the mixed MBs were then washed twice with 1 ml of 100 mM MES buffer, resuspended in 800 μL of MES buffer. Then an appropriate amount of 1.25 M EDC and RNA probe were added to make a total volume of 1 ml, rotating and incubated overnight at 4°C. The next day, after blocking the MBs twice with 1 ml of TT buffer (0.25 M Tris, 0.01% Tween -20, pH 8.0, 30 min per round of incubation), the MBs were resuspend with 1 ml of NAB. At last, ALP was added (according to the volume ratio of ALP:MBs = 1:10) to the MBs and rotating for 1 h. The MB-RNA-ALP complexes were washed twice with 1 ml of TT buffer and stored in TT buffer at 4°C.

For each test, 2 μL of primary MBs were added to the reaction solution (11.25 nM purified LwCas13a, 22.5 nM crRNA, 10 nM target RNA, 1 μL of murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from human PBMC), supplemented with NAB to 50 μL) and mixed on a roller mixer for 30 min. The activated LwCas13a cleaved the RNA probe and the ALP was released from the MB-RNA-ALP complexes. Non-target RNA or non-Cas13a protein served as a negative control (NC) in the detection system, and MBs complexes supplement 50 μL of NAB buffer served as background value. At the same time, MB-RNA-ALP complexes were incubated with RNase acting as a positive control, to confirm that the ALP could release from complexes and act as a signal molecule. After magnetic separation, 30 μL of the reaction solution and ALP substrate were incubated in a 96-well microplate format and the CL signal value was recorded using a microplate reader (SpectraMax M5) at 42°C for 15 min.

About 1 μL of mouse RNase inhibitor, 11.25 nM purified LwCas13a, 22.5 nM crRNA, 100 ng of background total human RNA and various concentrations of target RNA were mixed with MBs complexes and were further supplemented with NAB buffer to a final volume of 50 μL for HA RNA and NA RNA detection.

Improved detection assays were performed with RT-RAA nucleic acid amplification kit (Qitian Gene Biotechnology) and HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) as follows: 0.48 μM forward primer, 0.48 μM reverse primer, 14 mM magnesium acetate anhydrous, 12.5 μL mM rNTP, 2.5 μLT7 Mix, various concentrations of target RNA, 2 μL murine RNase inhibitor and 25 μL Basic E-mix to 50 μL final volume. After completing optimization of the RT-RAA amplification system for the cleaving system, MB-RNA-ALP complexes were added into the reaction solution (11.25 nM purified LwCas13a, 22.5 nM crRNA, 1 μL RNase inhibitor, 100 ng RNA purified from human PBMC and various concentration gradients of the RT-RAA amplification products of HA RNA) and replenished with NAB buffer 50 μL to a final volume of 50 μL for HA RNA and NA RNA detection.

H7N9 virus samples and other viruses samples, including seasonal influenza viruses (A/H1N1 and A/H3N2), 2009 swine-origin influenza virus A/H1N1, and avian influenza viruses (A/H5N1 and A/H9N1) donated by Jiangsu Provincial Center for Disease Prevention and Control were tested after amplification by RT-RAA. The testing was conducted using the same procedure as described in Section 2.6.

In our study, based on the structural features of PCO13-Twinstrep-SUMO-huLwCas13a-plasmid, the LwCas13a protein was purified by the SUMO enzyme. The molecular weight of LwCas13a containing the SUMO site and the marker protein was 155.2 kD, but it was 138.5 kD after digestion with SUMO enzyme. LwCas13a expressed in E. coli was analyzed by SDS-PAGE. Coomassie blue stained gel results showed that the IPTG significantly induced the production of Cas13a protein in E. coli (Figure 1A, lane 2). The bacterial solution after IPTG induction was collected, centrifuged, and the cell pellet was disrupted by sonication and centrifuged again. SDS-PAGE analysis was performed on disrupted cell supernatant and cell pellet. It was found that the LwCas13a was mainly present in the supernatant (Figure 1A, lane three and 4). Because the LwCas13a protein contains a His-tag, we used nickel ion affinity chromatography to adsorb water-soluble proteins. A protein without multiple consecutive histidine was eluted from the column by the wash buffer, while LwCas13a was still on the column, and then the SUMO site in LwCas13a was digested with SUMO enzyme, and the LwCas13a lost 6 × His was easily eluted from the column by lysis buffer, pointed by the black arrow (Figure 1B, lane 3). By this purification method a highly pure LwCas13a protein can be obtained.

RNase A can degrade RNA probes, so we used RNase A as a positive control to verify the RNase activity of LwCas13a. As shown in Figure 1C, the CL intensity increased with time and became stable after 10 min approximately. The CL intensity of positive sample (10 nM) was evidently higher than that of negative control (no target HA RNA or LwCas13a) and the blank, which indicated that Cas13a could non-specifically cleave RNA probes by binding the target RNA and its corresponding crRNA.

Given the non-specific absorption and steric hindrance effect, the density of probes on the surface of MBs may also affect the cleavage of RNA probes by Cas13a protein. About 1 mg MB and five different concentrations of RNA probes (0.1, 0.2, 0.4, 0.6 and 0.8 nM) were used to determine their effect on the CL intensity. We used 10 nM target RNA to activate Cas13a. Figure 2A showed that the CL intensity increased as the probe concentration increased. However, considering the positive judgment criterion, according to Figure 2A, there was a maximum S/N value at 0.4 nM, so 0.4 nM could be proposed as optimal concentration of RNA probes for the modification of MBs. The reaction temperature was investigated as a factor that may affect the cleaving efficiency of Cas13a. A temperature gradient (4, 18, 25, 37°C) was set to test the effect of the reaction temperature on the CL intensity. Figure 2B illustrated that as the temperature increased, the CL intensity increased, but the negative control and blank also increased. According to the change of S/N value, 25°C was selected as the optimal reaction temperature. Reaction time is another factor that affects the cleaving efficiency of Cas13a. A hybridization time gradient (10, 20, 30, 60, 90 min) was established to examine its effect on CL intensity. Figure 2C showed that in the range of 10–30 min, the CL intensity significantly increased with increased hybridization time and the CL intensity gradually stabilized after 30 min, but the negative control also increased with time. Therefore, 30 min was considered to be the best reaction time.

The schematic of Cas13a-MB-CL detection system was illustrated in Figure 3A. The detection limit of Cas13a-MB-CL detecting HA RNA was 100 p.m. (Figure 3C). Besides, Cas13a-MB-CL was used to detect NA RNA, having the same detection limit (Supplementary Figure S1). RT-RAA assay is a newly developed technology which can react at constant temperature and within a short reaction time (30 min) (Wang et al., 2022; Wu et al., 2022). Hence, we combined the CRISPR/Cas system with RT-RAA to improve sensitivity to detect H7N9. The results in Supplementary Figure S2 showed that the RT-RAA amplification system (RT-RAA-Cas13a-MB-CL) did have an effect on the cleaving system, and the CL intensity of positive group was gradually enhanced from the original multiplier products to the 16-fold dilution of products, weakened in 32-fold dilution of products. Therefore, the products of the 16-fold dilution were selected as the optimal optimization condition to be added to the cleaving system (Supplementary Figure S2). The proposed RT-RAA-Cas13a-MB-CL has a much better detection sensitivity compared to Cas13a-MB-CL assay in theory. Then, we investigated CL signals between the two methods in response to different concentrations of HA RNA (Figures 3B,C) and NA RNA (Supplementary Figure S1). In Figure 3B, the CL response signals of 1 nM HA RNA from RT-RAA-Cas13a-MB-CL and Cas13a-MB-CL assay were determined to be 6.56 and 3.89 compared to blank controls, respectively, suggesting an efficient signal gain in RT-RAA-Cas13a-MB-CL. And more importantly, RT-RAA-Cas13a-MB-CL was able to decrease the limit of detection (LOD) by approximately three orders of magnitude compared with the Cas13a-MB-CL assay (Figure 3C). Here, F/F0 was defined as the CL response signal, where F and F0 are the CL intensities of each assay with and without the presence of HA/NA RNA, respectively. Moreover, the LOD of RT-RAA-Cas13a-MB-CL is one to two orders of magnitude lower than that of Cas13a-fluorescence based RNA assays (Abudayyeh et al., 2016). To calculate the LOD of RT-RAA-Cas13a-MB-CL, a linear relationship between the CL response signal and the logarithmic value of H7N9 RNA concentration was obtained in the concentration range from 100 fM to 1 nM, with a LOD of 19.7 fM (3S/N, R ² = 0.98) (Choengchan et al., 2006; nanocomposite, 2020) (Figure 3D, Supplementary Figure S3).

The clinical performance of RT-RAA-Cas13a-MB-CL was evaluated using H7N9 virus samples donated by Jiangsu Provincial Center for Disease Prevention and Control. And the CL values of HA and NA in the two clinical samples were greater than that of negative controls (Figures 4A,B), showing a superior sensitivity and specificity.

In addition, the detection of H7N9 RNAs by the RT-RAA-Cas13a-MB-CL should be sequence-specific, so we tested seasonal influenza viruses (A/H1N1 and A/H3N2), 2009 swine-origin influenza virus A/H1N1, and avian influenza viruses (A/H5N1 and A/H9N1) donated by Jiangsu Provincial Center for Disease Prevention and Control (Sup. Table 1). The CL detection signals of H7N9 virus samples were significantly higher compared to other virus samples (Figure 5, Supplementary Figure S4). Above all, the proposed method had high specificity in the detection of H7N9 RNAs. Compared with RT-qPCR, the specificity of this method was comparable or even much better. Although slightly less sensitive than RT-qPCR, the entire detection process of this method can be completed in 30 min, while RT-qPCR needs more than 1 hour.