Sep 08, 2020

Public workspaceITP-CRISPR detection of SARS-CoV-2 RNA

DOI
dx.doi.org/10.17504/protocols.io.bkxnkxme
  • Ashwin Ramachandran1,
  • Diego A. Huyke2,
  • Eesha Sharma3,
  • Malaya K. Sahoo4,
  • ChunHong Huang4,
  • Niaz Banaei4,5,
  • Benjamin A. Pinsky4,5,
  • Juan G. Santiago2
  • 1Department of Aeronautics & Astronautics, Stanford University, California, USA 94305;
  • 2Department of Mechanical Engineering, Stanford University, California, USA 94305;
  • 3Department of Biochemistry, Stanford University, Stanford, California, USA 94305;
  • 4Department of Clinical Pathology, Stanford University, Stanford, California, USA 94305;
  • 5Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, California, USA 94305
  • XPRIZE Rapid Covid Testing
  • ITP-CRISPR detection of SARS-CoV-2 RNA
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DOI: dx.doi.org/10.17504/protocols.io.bkxnkxme
Protocol Citation: Ashwin Ramachandran, Diego A. Huyke, Eesha Sharma, Malaya K. Sahoo, ChunHong Huang, Niaz Banaei, Benjamin A. Pinsky, Juan G. Santiago 2020. ITP-CRISPR detection of SARS-CoV-2 RNA. protocols.io https://dx.doi.org/10.17504/protocols.io.bkxnkxme
Manuscript citation:
https://www.biorxiv.org/content/10.1101/2020.05.21.109637v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: September 06, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41678
Keywords: CRISPR-diagnostics, Microfluidics, Electrokinetics, SARS-CoV-2, Nucleic acid test, COVID-19, RT-LAMP, Isotachophoresis (ITP) ,
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Abstract
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.
Materials
Materials list:

Reagent or ConsumableSupplierCatalog #Amount
WarmStart® LAMP Kit (DNA & RNA)New England BiolabsE1700S1.25 mL
Wash Solution 1CustomN/A10 mL
Wash Solution 2CustomN/A10 mL
Wash Solution 3Custom N/A10 mL
Wash Solution 4CustomN/A10 mL
EnGen® Lba Cas12a (Cpf1)New England BiolabsM0653T2000 pmol
LAMP primer N gene (10x mix)Elim BiosciencesN/A10 mL
LAMP primer E gene (10x mix)Elim BiosciencesN/A10 mL
LAMP primer RNase P gene (10x mix)Elim BiosciencesN/A10 mL
E gene gRNA (10 x)IDTN/A5 mL
N gene gRNA (10 x)IDTN/A5 mL
RNase P gene gRNA (10 x)IDTN/A5 mL
Reporter ssDNA (10 x)IDTN/A10 mL
Nuclease free waterThermo Fisher10977015500 mL
Lysing buffer (10 x)CustomN/A10 mL
LE1 extraction CustomN/A50 mL
LE1 elutionCustomN/A50 mL
TE1 (10 x)CustomN/A5 mL
LE2 Custom N/A50 mL
TE2CustomN/A10 mL
Equipment list:

EquipmentSupplierModel
MicroscopeNikonTE200
sCMOS camera + softwareHamamatsu ORCA-Flash4.0
SourcemeterKeithley2410
Blue LED light souceThorlabsM470L3
Microfluidic chipCaliper life sciencesNS12AZ
WaterbathCustomN/A
Vacuum pumpCustomN/A
Additional equipment and consumables:
  • Pipette (P10, P20 and P200)
  • Pipette tips (10 µL, 20 µL and 200 µL)

LAMP primerSequence (5'-3')
N-gene F3AAC ACA AGC TTT CGG CAG
N-gene B3GAA ATT TGG ATC TTT GTC ATC C
N-gene FIPTGC GGC CAA TGT TTG TAA TCA GCC AAG GAA ATT TTG GGG AC
N-gene BIPCGC ATT GGC ATG GAA GTC ACT TTG ATG GCA CCT GTG TAG
N-gene LFTTC CTT GTC TGA TTA GTT C
N-gene LBACC TTC GGG AAC GTG GTT
E-gene F3CCG ACG ACG ACT ACT AGC
E-gene B3AGA GTA AAC GTA AAA AGA AGG TT
E-gene FIPACC TGT CTC TTC CGA AAC GAA TTT GTA AGC ACA AGC TGA TG
E-gene BIPCTA GCC ATC CTT ACT GCG CTA CTC ACG TTA ACA ATA TTG CA
E-gene LFTCG ATT GTG TGC GTA CTG C
E-gene LBTGA GTA CAT AAG TTC GTA C
RNaseP POP7 F3TTG ATG AGC TGG AGC CA
RNaseP POP7 B3CAC CCT CAA TGC AGA GTC
RNaseP POP7 FIPGTG TGA CCC TGA AGA CTC GGT TTT AGC CAC TGA CTC GGA TC
RNaseP POP7 BIPCCT CCG TGA TAT GGC TCT TCG TTT TTT TCT TAC ATG GCT CTG GTC
RNaseP POP7 LFATG TGG ATG GCT GAG TTG TT
RNaseP POP7 LBCAT GCT GAG TAC TGG ACC TC
qPCR primerSequence (5'-3')
E_Sarbeco_F1ACA GGT ACG TTA ATA GTT AAT AGC GT
E_Sarbeco_R2ATA TTG CAG CAG TAC GCA CAC A
E_Sarbeco_P15 -FAM/ACA CTA GCC ATC CTT ACT GCG CTT CG/3 -BHQ-1
RP-FAGA TTT GGA CCT GCG AGC G
RP-RGAG CGG CTG TCT CCA CAA GT
RP-P5 -FAM/TTC TGA CCT GAA GGC TCT GCG CG/3 -BHQ-1
gRNASequence (5'-3')
E geneUAA UUU CUA CUA AGU GUA GAU GUG GUA UUC UUG CUA GUU AC
N geneUAA UUU CUA CUA AGU GUA GAU CCC CCA GCG CUU CAG CGU UC
RNase PUAA UUU CUA CUA AGU GUA GAU AAU UAC UUG GGU GUG ACC CU
Template and reporterSequence (5'-3')
ssDNA reporter/56-FAM/TTATT/3IABkFQ/
Primers and guide RNA sequences used in this assay
Before start
Wear appropriate PPE including gloves, lab coat, goggles.
Raw NP swab samples must be handled according to BSL-2 safety level or higher.
Chip preparation before testing
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.

Note
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
ITP extraction of total nucleic acids
Mix Amount25 µL of raw NP swab sample in VTM with Amount3 µL of 10 x Lysing buffer, pipette mix, and incubate at Temperature62 °C for Duration00:02:00 in a water bath.

Add Amount3 µL of 10x TE1 to the lysate, pipette mix, and load Amount20 µL of this mix into the Reservoir 1 of the chip
Figure 1. Chip layout and loading procedure.

Load Amount20 µL of LE1 in reservoirs 2 and 4 each, as shown in Figure 1, and apply vacuum using a vacuum pump for Duration00:00:15 at reservoir 3 till the main channel is completely filled as depicted in Figure 1.

Empty Reservoir 3 of any residual liquid, and load Amount20 µL of LE1 elution buffer in it.

Apply Amount1000 V voltage between reservoirs 1 and 3 as shown in Figure 1 for approximately Duration00:03:00 . 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.
Figure 2. Visualization of ITP peak during extraction and elution of total nucleic acids from raw NP swab samples

Pipette out Amount20 µL of elution volume containing extracted nucliec acids into an Eppendorf tube and place on ice until further use.

RT-LAMP for N, E, and RNase P genes
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.
ReagentVolume (per E gene reaction)Volume (per N gene reaction)Volume (per RNase P gene reaction)
WarmStart LAMP 2X Master Mix10 µL10 µL10 µL
LAMP Primer Mix (10X)2 µL (E)2 µL (N)2 µL (RNase P)
Template from Eluate6 µL6 µL6 µL
Nuclease free water2 µL2 µL2 µL
Total Volume20 µL20 µL20 µL
LAMP primer component10x concentration1x concetration
FIP16 μM1.6 μM
BIP16 μM1.6 μM
F32 μM0.2 μM
B32 μM0.2 μM
LOOP F8 μM0.8 μM
LOOP B8 μM0.8 μM
LAMP 10x primer mix for N, E, and RNase P genes
Incubate the above mixtures at Temperature62 °C for Duration00:20:00 to Duration00:30:00 and perform LAMP for N, E and RNase P genes in independent tubes. Place tubes on ice after LAMP.

ITP-CRISPR detection of cDNA of LAMP amplicons
ITP-CRISPR detection of cDNA of LAMP amplicons
Prepare 10x RNP mix as follows for each N, E, and RNase P genes

ReagentVolume (per E gene reaction)Volume (per N gene reaction)Volume (per RNase P gene reaction)
NEBuffer 2.1 (10x)2 uL2 uL2 uL
NEB LbCas12a (100 uM)0.2 uL0.2 uL0.2 uL
gRNA (10 x)17.8 uL (E)17.8 uL (N)17.8 uL (RNase P)
total volume20 uL20 uL20 uL
Incubate the mixtures in Temperature37 °C forDuration00:30:00 and then place on ice.

Add Amount3 µL of the 10x RNP mixes prepared above to Amount27 µL of LE2, independently for N, E and RNase P genes, and then place the three mixtures on ice.

Add Amount2 µL of 10x reporter ssDNA to Amount18 µL of TE2.

LoadAmount20 µL of LE2 in reservoirs 5 and 8 (as per Figure 1).

Mix Amount2 µL of LAMP amplicon for N gene with Amount18 µL of the RNP+LE2 mix in Step 11 corresponding to the N gene, and load this Amount20 µL in reservoir 6.
Note
Note that a lower volume of Amount0.5 µL 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 Duration00:00:01 after loading RNP+LE2 , and then load the remainder of reservoir 6 with Amount19.5 µL of LE2.



Apply vaccum for Duration00:00:10 at reservoir 7 using the vacuum pump. Clear reservoir 7 of any residual liquid.

Load Amount20 µL of the mix prepared in Step 12 to reservoir 7.

Apply Amount4 μA 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.


Expected result
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.

Perform wash step 1 and repeat steps 14 to 18 for the E gene and Rnase P gene.
Note
Future iterations of this protocol will involve multiplexed detection of the N, E and RNase P genes on a single chip.

Expected result for positive and negative detection of SARS-CoV-2 samples.
Expected result
Figure 3. Raw experimental visualization of the ITP peak fluorescence intensity
Figure 4. Measured kinetic profiles of fluorescence and end-point fluorescence readout for the N, E and RNase P genes. LOD is 10 copies per uL



Note
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.