TITLE: ITP-CRISPR detection of SARS-CoV-2 RNA
AUTHORS: Ashwin Ramachandran, Diego A. Huyke, Eesha Sharma, Malaya K. Sahoo, ChunHong Huang, Niaz Banaei, Benjamin A. Pinsky, Juan G. Santiago

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA).This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter non-specifically cleaves single-stranded DNA reporter molecules labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. We also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification (LAMP) and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in about 30 min for both contrived and clinical nasopharyngeal swab samples. Our goal is to use validated LAMP primers and gRNAs and implement them on our ITP-CRISPR microfluidic platform. The on-chip electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays and makes the technology amenable to automation and point-of-care applications.</div></div>

[STEPS]
?. [Chip preparation before testing]
Rinse the NS12AZ channel in the following order: Wash solution 1 for 2 min, Wash solution 2 for 2 min, Wash solution 3 for 2 min, Wash solution 2 for 2 min, Wash solution 4 for 2 min, and Wash solution 3 for 2 min. Between each rinse step, dry the channel using vacuum.
In the current protocol, the NS12AZ caliper chips can be reused for multiple samples. Wash proceduce described here ensures no cross contamination. Future iterations of the protocol will invovle the use of pre-prepared disposable chips for which this wash step can be skipped.
?. [ITP extraction of total nucleic acids]
Mix  of raw NP swab sample in VTM with  of 10 x Lysing buffer, pipette mix, and incubate at for  in a water bath.
25 µl
3 µl
62 °C
?. [ITP extraction of total nucleic acids]
Add  of 10x TE1 to the lysate, pipette mix, and load  of this mix into the Reservoir 1 of the chip
3 µl
20 µl
?. [ITP extraction of total nucleic acids]
Load  of LE1 in reservoirs 2 and 4 each, as shown in Figure 1, and apply vacuum using a vacuum pump for at reservoir 3 till the main channel is completely filled as depicted in Figure 1.
20 µl
?. [ITP extraction of total nucleic acids]
Empty Reservoir 3 of any residual liquid, and load  of LE1 elution buffer in it.
20 µl
?. [ITP extraction of total nucleic acids]
Apply  voltage between reservoirs 1 and 3 as shown in Figure 1 for approximately . Visualize ITP peak containing total nucleic acids using the microscope and a camera. Turn off voltage when the ITP peak reaches the elution reservoir 3.
1000
?. [ITP extraction of total nucleic acids]
Pipette out  of elution volume containing extracted nucliec acids into an Eppendorf tube and place on ice until further use.
20 µl
?. [RT-LAMP for N, E, and RNase P genes]
Prepare the following RT-LAMP mixtures each for N, E, and RNase P genes. Template is obtained from the eluate in the previous step.  ABCD1ReagentVolume (per E gene reaction)Volume (per N gene reaction)Volume (per RNase P gene reaction)2WarmStart LAMP 2X Master Mix10 µL10 µL10 µL3LAMP Primer Mix (10X)2 µL (E)2 µL (N)2 µL (RNase P)4Template from Eluate6 µL6 µL6 µL5Nuclease free water2 µL2 µL2 µL6Total Volume20 µL20 µL20 µL
ABCD1ReagentVolume (per E gene reaction)Volume (per N gene reaction)Volume (per RNase P gene reaction)2WarmStart LAMP 2X Master Mix10 µL10 µL10 µL3LAMP Primer Mix (10X)2 µL (E)2 µL (N)2 µL (RNase P)4Template from Eluate6 µL6 µL6 µL5Nuclease free water2 µL2 µL2 µL6Total Volume20 µL20 µL20 µL
?. [RT-LAMP for N, E, and RNase P genes]
Incubate the above mixtures at  for  to  and perform LAMP for N, E and RNase P genes in independent tubes. Place tubes on ice after LAMP.
62 °C
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Prepare 10x RNP mix as follows for each N, E, and RNase P genes  ABCD1ReagentVolume (per E gene reaction)Volume (per N gene reaction)Volume (per RNase P gene reaction)2NEBuffer 2.1 (10x)2 uL2 uL2 uL3NEB LbCas12a (100 uM)0.2 uL0.2 uL0.2 uL4gRNA (10 x)17.8 uL (E)17.8 uL (N)17.8 uL (RNase P)5total volume20 uL20 uL20 uLIncubate the mixtures in  for and then place on ice.
ABCD1ReagentVolume (per E gene reaction)Volume (per N gene reaction)Volume (per RNase P gene reaction)2NEBuffer 2.1 (10x)2 uL2 uL2 uL3NEB LbCas12a (100 uM)0.2 uL0.2 uL0.2 uL4gRNA (10 x)17.8 uL (E)17.8 uL (N)17.8 uL (RNase P)5total volume20 uL20 uL20 uL
37 °C
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Add  of the 10x RNP mixes prepared above to  of LE2, independently for N, E and RNase P genes, and then place the three mixtures on ice.
3 µl
27 µl
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Add  of 10x reporter ssDNA to  of TE2.
2 µl
18 µl
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Load of LE2 in reservoirs 5 and 8 (as per Figure 1).
20 µl
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Mix  of LAMP amplicon for N gene with  of the RNP+LE2 mix in Step 11 corresponding to the N gene, and load this  in reservoir 6.
2 µl
18 µl
20 µl
Note that a lower volume of    for RNP+LE2 containing CRISPR regaents can alternately be used in reservoir 6 to minimize reagent consumption. In this case, apply vacuum at reservoir 7 (step 15) for only   after loading RNP+LE2 ,  and then load the remainder of reservoir 6 with   of LE2.
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Apply vaccum for at reservoir 7 using the vacuum pump. Clear reservoir 7 of any residual liquid.
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Load  of the mix prepared in Step 12 to reservoir 7.
20 µl
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Apply of current between reservoirs 6 and 7 (as per figure 1) and monitor the fluorescence of the ITP peak using a custom microscope-LED-camera system.
4
A positive sample shows a rapid increase in fluorescence signal of the ITP peak and a value above the threshold value. The threshold is determined using prior calibration experiments. A negative sample has low or minimal increase in fluorescence signal of the ITP peak.
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Perform wash step 1 and repeat steps 14 to 18 for the E gene and Rnase P gene.
Future iterations of this protocol will involve multiplexed detection of the N, E and RNase P genes on a single chip.
?. [ITP-CRISPR detection of cDNA of LAMP amplicons]
Expected result for positive and negative detection of SARS-CoV-2 samples.
Current protocol is optimized for testing one sample per run. Future version of our protocol will multiplex 96 target reactions and samples in a single run.
?. [RT-LAMP for N, E, and RNase P genes]
ABC1LAMP primer component10x concentration1x concetration2FIP16 μM1.6 μM3BIP16 μM1.6 μM4F32 μM0.2 μM5B32 μM0.2 μM6LOOP F8 μM0.8 μM7LOOP B8 μM0.8 μM
ABC1LAMP primer component10x concentration1x concetration2FIP16 μM1.6 μM3BIP16 μM1.6 μM4F32 μM0.2 μM5B32 μM0.2 μM6LOOP F8 μM0.8 μM7LOOP B8 μM0.8 μM