AIOD-CRISPR assay system. As shown in Figure 1A, the AIOD-CRISPR assay system uses a pair of Cas12a-crRNA complexes generated by two individual crRNAs to bind two different sites which are close to the recognition sites of primers in the target sequence. The Cas12a- crRNA complexes are first prepared prior to being adding into the reaction solution containing RPA primers, ssDNA-FQ reporters, recombinase, single-stranded DNA binding protein (SSB), strand-displacement DNA polymerase, and target sequences. When incubating the AIOD- CRISPR reaction system in one pot at ~37°C, the RPA amplification is initiated and exposes the binding sites of the Cas12a-crRNA complexes due to the strand displacement. On one hand, when the Cas12a-crRNA complexes bind the target sites, the Cas12a endonuclease is activated and cleaves the ssDNA-FQ reporters, generating strong fluorescence signals. On the other hand, the amplified products generated during the RPA continuously trigger CRISPR-Cas12a-based collateral cleavage activity. Previous studies 14, 17 have demonstrated that the collateral cleavage activity of the CRISPR-Cas12a system is independent of target strand cleavage. Therefore, target sequences for our AIOD-CRISPR assay are not limited by the Cas12a’s protospacer adjacent motif (PAM) 22.
To systematically evaluate our AIOD-CRISPR assay system, we prepared and tested eight reaction systems (reactions # 1–8) with various components (Figure 1B (i)).. The ssDNA-FQ reporter was a 5 nt oligonucleotide (5’-TTATT–3’) labelled by 5’ 6-FAM (Fluorescein) fluorophore and 3’ Iowa Black® FQ quencher. After incubation at 37°C for 40 min, only reaction # 5 containing target nucleic acid sequence, dual crRNAs, Cas12a, and RPA reaction mixture produced super-bright fluorescence signal (Figure 1B (i)),, which could be directly visualized under a blue LED or UV light illuminator. Surprisingly, even under ambient light conditions without excitation, a color change from orange-yellow to green was directly observed in the reaction tube # 5 by naked eyes. To further verify the specificity of the generated fluorescence signal, the assay products (self-probed fluorescence reporters) were subjected to denaturing polyacrylamide gel electrophoresis (PAGE). As shown in Figure 1B(ii), a strong band with shorter DNA size was observed only in the lane of reaction # 5, which resulted from the cleaved ssDNA-FQ reporters with strong fluorescence signal. In comparison, for other reaction systems, only weak bands with relatively longer DNA sizes were observed in their corresponding lanes, which may be attributed to fluorescence quench of the intact uncut ssDNA-FQ reporters. In addition, in real-time fluorescence curves, only reaction # 5 showed a significantly increased fluorescence signal that saturated at 13 min (Figure 1B(iii)).. Thus, these results show that our AIOD-CRISPR assay provides a simple, rapid, one-pot approach for target-specific nucleic acid detection.
Since a previous study reported that RPA amplification reaction is initiated after adding MgOAc,23 we are interested in knowing if nucleic acid amplification is efficiently initiated at room temperature during sample preparation in our AIOD-CRISPR assay system. We prepared two AIOD-CRISPR solutions (one positive and one negative) and allowed them to remain at room temperature for 10 min. As shown in Figure S1, no significant fluorescence change between positive and negative samples was observed in the AIOD-CRISPR systems at room temperature. In comparison, there was an obvious fluorescence increase after just one-minute incubation at 37 °C (Figure S1).. Eventually, the fluorescence signal was saturated after 15-min incubation at 37 °C. Therefore, our AIOD-CRISPR assay system is mainly triggered after reaction temperature is elevated to ~37°C.
Optimization of AIOD-CRISPR assay. Collateral cleavage of the ssDNA-FQ reporters by the Cas12a nuclease is triggered by the binding of crRNA to target sites.13, 14 Here, we hypothesize that more binding opportunities can increase the collateral cleavage activity and eventually improve the detection sensitivity. To test our hypothesis, we designed a pair of crRNAs to respectively recognize two different target sites in our AIOD-CRISPR assay. A pUCIDT-AMP plasmid containing 300 bp HIV–1 p24 gene cDNA (p24 plasmid) was used as the target and three different design strategies for primers and crRNAs were investigated (Figure 2A).. As shown in Figure 2B, the AIOD-CRISPR with dual crRNAs (crRNA1+crRNA2) showed slightly higher fluorescence signals compared to that with single crRNA2, but much better than that with single crRNA1. In addition, doubling the amount of either crRNA1 or crRNA2 did not benefit the detection efficiency. Furthermore, we evaluated and compared the detection sensitivity of the AIOD-CRISPR system with dual crRNAs and single crRNA. As shown in Figure 2C and D, the AIOD-CRISPR with dual crRNAs was able to consistently detect as low as 1.2 copies of the p24 plasmid templates with improved fluorescence, while the AIOD- CRISPR assay with single crRNA2 did not. Thus, by introducing dual crRNAs into the AIOD- CRISPR assay, it does not only increase fluorescence signals, but also improve the detection sensitivity.
We further optimized ssDNA-FQ reporters in our AIOD-CRISPR assay because the reporter concentration plays a crucial role in fluorescence readout. As shown in Figure S2A, the higher the concentration of the ssDNA-FQ reporters, the stronger the fluorescence signal and the shorter the threshold time. As to threshold time and visual detection, the minimal concentration for saturated values was 4 μM (Figure S2B-S2D).. Collateral cleavage efficiency of the activated Cas12a nuclease represents an ability to cut ssDNA-FQ reporters around it.13, 14 Thus, increasing the ssDNA-FQ reporter concentration can improve the fluorescence signals. In addition, we also investigated the effect of the primer concentration on the AIOD-CRISPR assay. As shown in Figure S3, the optimal concentration of the primers was 0.32 μM. Together, introducing dual crRNAs with an increased ssDNA-FQ reporter concentration enables highly efficient AIOD-CRISPR assay.
HIV–1 detection by AIOD-CRISPR assay. To investigate the sensitivity of the AIOD-CRISPR assay for HIV–1 DNA detection, we first applied the optimized AIOD-CRISPR assay to detect various copies of HIV–1 p24 plasmid templates (from 1.2× 100 to 1.2× 105 copies). As shown in Figure 3A, the AIOD-CRISPR could consistently detect as low as 1.2 copies HIV–1 p24 plasmid DNA in both real-time and endpoint visual detection, which was further verified by the denaturing PAGE. Although incubated for 40 min, the AIOD-CRISPR assay could detect and identify 1.2 copies of HIV–1 DNA in just 1-min incubation based on the endpoint fluorescence intensity (Figure S4),, which shows that our AIOD-CRISPR assay provides a super-fast (few minutes) and ultrasensitive (several copies) detection of nucleic acids.
Next, we applied the AIOD-CRISPR assay to detect HIV–1 RNA sequence by adding Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, namely reverse transcription AIOD- CRISPR (RT-AIOD-CRISPR). The HIV RNA target is a 1057-nt fragment of gag (p24 included) gene prepared using T7 promotor-tagged RT-PCR and T7 RNA polymerase-based transcription (Figure S5A).. The detection region of the AIOD-CRISPR assay was further verified by Sanger sequencing (Figure S5B).. To achieve highly sensitive HIV–1 RNA detection, we optimized the AMV concentration. As shown in Figure S5C, the RT-AIOD-CRISPR performed the highest efficiency with 0.32 U/μL AMV when incubated at 37°C. Furthermore, we investigated the detection sensitivity of our RT-AIOD-CRISPR assay using 1.1× 100,1.1× 101, 1.1× 103, and 1.1× 105 copies of HIV–1 gag RNA templates. Figure 3B showed that the RT-AIOD-CRISPR assay was able to consistently detect 11 copies of HIV–1 RNA targets in both real-time and endpoint fluorescence visual detections. However, visual detection without excitation light can only identify 1.1× 105 copies of HIV–1 RNA targets. Additionally, the RT- AIOD-CRISPR’s sensitivity was further confirmed by denaturing PAGE analysis (Figure 3B)..
In addition to detecting artificial HIV–1 gag RNA, we also evaluated the RT-AIOD-CRISPR’s performance using HIV–1 RNA extracted from human plasma samples. As shown in Figure 3C, real-time RT-AIOD-CRISPR assay was able to detect 500 copies of HIV viral RNA within less than 20 min. However, visual RT-AIOD-CRISPR detection took relatively long incubation time (up to 90 min) to achieve the similar sensitivity (Figure S6A).. The reduced sensitivity of the RT-AIOD-CRISPR for extracted HIV RNA detection may be attributed to either potential inhibitors in the extracts or RNA degradation during extraction. Despite this, the real-time AIOD-CRISPR assay showed a comparable sensitivity compared to real-time RT-PCR assay (Figure S6B)..
SARS-CoV–2 detection by AIOD-CRISPR assay. As shown in Figure 4A, a pUCIDT-AMP plasmid containing 316 nt SARS-CoV–2 N gene cDNA (N plasmid) was first prepared as the target to develop our AIOD-CRISPR assay. Figure 4B shows that our AIOD-CRISPR assay could detect 1.3 copies of SARS-CoV–2 N plasmids in both real-time and visual detections, offering a rapid and nearly single-molecule level sensitive detection. To evaluate the detection specificity, we tested our AIOD-CRISPR assay using commercially available control plasmids containing the complete N gene from SARS-CoV–2 (SARS-CoV–2 _PC, Catalogue # 10006625, IDT), SARS (SARS-CoV_PC, Catalogue # 10006624, IDT), and Middle East respiratory syndrome (MERS) (MERS-CoV (Middle East respiratory syndrome coronavirus)_PC, Catalogue # 10006623, IDT), as well as the Hs_RPP30 control (Hs_RPP30_PC, Catalogue # 10006626, IDT) with a portion of human RPP30 gene. Figure 4C shows that only the reaction with SARS-CoV–2_PC had the positive signal in both real- time and visual detections, demonstrating that our developed AIOD-CRISPR assay possesses high specificity without cross reactions for non-SARS-CoV–2 targets.
Next, we used T7 promotor-tagged PCR and T7 RNA polymerase to prepare SARS-CoV–2 N gene RNA sequences to develop the RT-AIOD-CRISPR assay (Figure S7A).. The detection region of the RT-AIOD-CRISPR was verified by Sanger sequencing (Figure 7B).. As shown in Figure 4D, the RT-AIOD-CRISPR assay could consistently detect down to 4.6 copies of SARS-CoV–2 N RNA targets in both real-time and visual detections. In addition, Figure 4E shows that the endpoint fluorescence intensity after 1 min RT-AIOD-CRISPR reaction was able to identify 4.6 copies of SARS-CoV–2 N RNA. Therefore, our AIOD-CRISPR assay provides an ultrarapid, highly sensitive and specific method for SARS-CoV–2 detection.
To further demonstrate its point of care diagnostic application, we used a low-cost hand warmer (~$ 0.3 per bag) as the incubator of our AIOD-CRISPR assay and detect COVID–19 patient samples. As shown in Figure 6A, the AIOD-CRISPR assay tubes were directly placed on an air-activated hand warmer without need for any electric incubator. The endpoint fluorescence result can be observed by the naked eye under LED light. Figure 6B shows that two SARS-CoV–2-positive samples incubated in the hand warmer bag were visually detected and identified within as short as 20 min. The longer the incubation time, the stronger the fluoscence signal of the positive samples. Additionally, a similar result was achieved through analyzing the green value of the fluorescence images using the ImageJ software (Figure 6C).. Therefore, our AIOD-CRISPR method provides a simple, rapid and visual approach for SARS- CoV–2 detection and has the potential to develop an instrument-free point of care diagnostics of the COVID–19.