All-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay protocol for SARS-CoV-2 detection

This protocol presents the all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay to ultra-sensitively and visually detect SARS-CoV-2. The procedure of AIOD-CRISPR assay typically consists of three parts including sample preparation, AIOD-CRISPR reaction, and uorescence detection. Sample preparation involves the synthetic RNA preparation and the nucleic acid extraction from SARS-CoV-2 samples. The prepared nucleic acids were then added into the AIOD-CRISPR reaction systems as templates, followed by incubation at 37°C for 20-40 min. After incubation, visual detection was immediately conducted by placing the tubes in a portable LED blue transilluminator (Maestrogen UltraSlim) or the ChemiDoc™ MP Imaging System (Bio-Rad) with its built-in UV channel. In addition to endpoint visual detection, real-time uorescence detection was also available for AIOD-CRISPR assay. This protocol is helpful for applying AIOD-CRISPR assay to rapid, sensitive, one-pot point-of-care SARS-CoV-2 detection.

2. Pipette 20 μL of the PCR products for 2% agarose gel electrophoresis analysis (120 V and 45 min). Then cut the band at the size of about 336 bp, followed by gel extraction using PureLink™ Quick Gel Extraction Kit. Further con rm the sequence by shipping the puri ed PCR products to Genewiz ® for Sanger sequencing.
3. Incubate the solution containing 8 μL of 5× T7 Transcription Buffer, 3 μL each of 100 mM rNTPs, 4 μL of the Enzyme Mix with T7 RNA polymerase, 16 μL of the gel-extracted PCR products at 37°C for 4 h. The reagents are from the RiboMAX™ Large Scale RNA Production Systems-T7 Kit.
4. Treat the transcription products using the DNase (from the TURBO DNA-free TM Kit) to degrading the DNA.
5. Purify the resulting RNA using the RNeasy @ MinElute TM Cleanup Kit.
6. Determined the purity and concentration of the collected nucleic acid using NanoDrop™ One/One C Microvolume UV-Vis Spectrophotometry. 3. Pipette 2.5 μL of the synthetic RNA or the extracted nucleic acid solutions into the prepared Component A, followed by adding 3.25 of the prepared Component B. The assembled mixture is left at room temperature during the preparation of Component C. TIP: Thoroughly vortex the assembled mixture and invert the tube 3 times prior to slight centrifuging. 4. Prepare Component C by mixing 0.32 μL of 50 μM crRNA1, 0.32 μL of 50 μM crRNA2, 0.32 μL of 100 μM EnGen® Lba Cas12a (Cpf1), and 0.28 μL nuclease-free water. TIP: It is preferable to leave the mixture for about 10 min at room temperature for fully forming the crRNA-Cas12a complex. Prepare the mixture enough for at least 2 reactions for easy and accurate pipetting. For better performance, the Component C is recommended to be fresh prepared when testing. 5. Pipette 1.24 μL of the Component C into the assembled mixture in Step 3 to form the nal reaction system. TIP: Thoroughly vortex the reaction system and invert the tube 3 times prior to slight centrifuging.
6. Incubate the nal reaction system at 37°C for 20-40 min prior to uorescence detection.
Fluorescence detection · Take out the tubes after the AIOD-CRISPR reaction and place them in the LED blue transilluminator or the Imaging System for visual uorescence detection. · Real-time uorescence detection can be achieved by incubating the AIOD-CRISPR reaction systems in the CFX96 Touch™ Real-Time PCR Detection System.

Time Taken
For real sample testing · Extract RNA from samples: at least 20 min. The actual time depends on sample size, manual or fully automated operation (e.g. the QIAcube).

·
Prepare the AIOD-CRISPR reaction system: at least 5 min. Component A, B, and C can be prepared as stock solutions and kept at -20°C until use. The actual time also depends on sample size. · Incubate the AIOD-CRISPR solution: 20-40 min.
· Run uorescence detection and visually judge the test results: 1-2 min.
Totally, an AIOD-CRISPR assay can be nished as short as 46 min from sample preparation to reporting results.