TITLE: Fluorescent-Reporter Based Assay
AUTHORS: Piyush Jain, Long T  Nguyen, Santosh Rananaware

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The CRISPR-Enhance SARS-CoV-2 detection kit has been designed to detect fragments of the Nucleocapsid (“N”) gene and Envelope gene (E) of SARS-CoV-2. An included third target is the human RNase P POP7 gene (“RP”) which serves as a control for the extraction of the clinical sample in the absence of a positive SARS-CoV-2 result. Amplification can be performed using a heat block, and CRISPR complex activation and reporter cleavage can be run in a standard microplate reader capable of fluorescence detection. The entire reaction from RT-LAMP amplification to CRISPR-based detection of the target analytes can be performed in approximately one hour.</div><div class = "text-block">The CRISPR-Enhance kit comprises of two steps. Step one is a reverse transcriptase loop-mediated amplification (RT-LAMP) where targeted SARS-CoV-2 genomic RNA is reverse transcribed to DNA, and this DNA is amplified by a strand-displacing DNA polymerase. Step two is the transcription of the amplified DNA to activate the collateral cleavage activity of a CRISPR complex programmed to the target RNA sequence. This collateral activity results in cleavage of nucleic acid reporters, resulting in a fluorescent readout detected by a plate reader.</div></div>

[STEPS]
?. [RT-LAMP Master Mix Preparation]
Label a new  microcentrifuge tube for each target ( N, E and RP) and prepare a RT-LAMP Master Mix consisting of the WarmStart®Colorimetric LAMP 2X Master Mix with UDG.and the appropriate 10x Primer Mix using the recipe in Table 1 below. Make enough of each master mix for all samples to be tested and the necessary controls for each run.  ABC1Reagent NameVolume per reactionTotal Volume2WarmStart®Colorimetric LAMP 2X Master Mix with UDG12.5 µL12.5 µL x (N+1)310x Primer Mix (N, E or RP)2.5 µL2.5 µL x (N+1)4RNAse-Free Water5 µL5 µL x (N+1)5Total Volume20 µL20 µL x (N+1)N = number of extracted samples plus number of controls. Prepare enough for 1 extra (N + 1) sample to allow for overage during reaction set-up.
1.5 mL
ABC1Reagent NameVolume per reactionTotal Volume2WarmStart®Colorimetric LAMP 2X Master Mix with UDG12.5 µL12.5 µL x (N+1)310x Primer Mix (N, E or RP)2.5 µL2.5 µL x (N+1)4RNAse-Free Water5 µL5 µL x (N+1)5Total Volume20 µL20 µL x (N+1)
?. [RT-LAMP Amplification]
Label a strip tube () with the target name (e.g. N) and strip number corresponding to each sample.Add  of the RT-LAMP Master Mix from the previous step into one well for each sample and control to be amplified. Repeat for the remaining 2 targets using a new strip for each target (e.g. E or RP)Add of extracted RNA in each respective strip tube containing the RT-LAMP Master mix. Vortex the strip tube for  and spin down for in microcentrifuge with a  tube adaptor.  AB1ReagentVolume per reaction2RT-LAMP Master Mix20 μL3RNA Sample or Controls5 μL4Total Volume25 μLHeat the mixture at  for
0.2 mL
20 µl
5 µl
0.2 mL
AB1ReagentVolume per reaction2RT-LAMP Master Mix20 μL3RNA Sample or Controls5 μL4Total Volume25 μL
65 °C
?. [CRISPR-Cas Reaction Preparation]
Preheat a fluorescence microplate reader to .For each target tested label a  tube with the target name (e.g. N, E or RNASE-P) and “Cas Mix”. Prepare a CRISPR Cas Master Mix using the following recipe in Table 3 below, scaling as required for the number of assays to be run (one Cas assay for every RT-LAMP reaction). Incubate the mixture at  for Pulse vortex for and spin down for  in a microcentrifuge after all components are added.  ABC1Reagent NameVolume per ReactionVolume Total2NEB 2.1 Buffer1.2 μL1.2 μL x (N+1)33 μM crRNA (N or E or RP)0.8 μL0.8 μL x (N+1)43 μM lbCas12a0.4 μL0.4 μL x (N+1)5RNAse-Free Water9.6 μL9.6 μL x (N+1)6Total Volume12 μL12 μL x (N+1)N = number of extracted samples plus number of controls. Prepare enough for 1 extra (N + 1) sample to allow for overage during reaction set-up.In a separatemicrocentrifuge tube prepare the fluorecence reporter as per Table 4  ABC1Reagent NameVolume per ReactionVolume Total2FAM-FQ0.2 μL0.2 μL x (N*+1)3RNAse-Free Water25.8 μL25.8 μL x (N*+1)4Total Volume12 μL12 μL x (N*+1)N* = number of extracted samples multiplied by the total number of genes plus number of controls. Prepare enough for 1 extra (N* + 1) sample to allow for overage during reaction set-up
37 °C
1.5 mL
37 °C
ABC1Reagent NameVolume per ReactionVolume Total2NEB 2.1 Buffer1.2 μL1.2 μL x (N+1)33 μM crRNA (N or E or RP)0.8 μL0.8 μL x (N+1)43 μM lbCas12a0.4 μL0.4 μL x (N+1)5RNAse-Free Water9.6 μL9.6 μL x (N+1)6Total Volume12 μL12 μL x (N+1)
1.5 mL
ABC1Reagent NameVolume per ReactionVolume Total2FAM-FQ0.2 μL0.2 μL x (N*+1)3RNAse-Free Water25.8 μL25.8 μL x (N*+1)4Total Volume12 μL12 μL x (N*+1)
?. [CRISPR-Cas Detection]
Add 26 μL of the Fluorescent reporter Mix made in step 4 to each well of a 384 well-plate corresponding to the number of samples and controls for every gene (N, E or RP).Add 2 μL of the RT-LAMP product from step 3 to each well containing the fluorescent reporter.Add 12 μL of the CRISPR-Cas master mix to the wells containing the corresponding RT-Lamp product and fluorescent reporter.Seal the plate with an Optical sealOpen the plate reader software to create a read procedure. Set temperature to 37°C Select "Kinetic" run reading with a total read time of 30 min, and data collection intervals at 2.5 minsSave experiment in a designated place with an appropriate unique nameWhen plate loader extdends, load plate. Ensure plate is loaded in correct orientation. Read the data.
?. [Nucleic Acid Extraction]
Add   of patient sample to   of pre-aliquoted QuickExtract solution. Heat the above mixture at   for followed by  for .
10 µl
10 µl
65 °C
98 °C