For Step (2) detection of virus RNA using Cas13:
- Cleavage buffer (400mM Tris pH 7.4 in ddH2O). Prepared using 1M Tris pH 7.4
stock solution from Sigma Aldrich (T2194-100ML)
- SUPERase•In™ RNase Inhibitor (AM2694), ThermoFisher Scientific
- NxGen® T7 RNA Polymerase (30223-1), Lucigen
- Ribonucleotide Solution Set (N0450S), New England BioLabs
- Magnesium chloride solution (63069-100ML), Sigma Aldrich
- Storage Buffer for diluting Cas13 protein stock (combine 2.5 mL of 1M Tris pH
7.4, 6 mL of 5M NaCl, 2.5 mL of glycerol, and 100 μL of 1M DTT, and 38.9 mL
ddH2O)
- LwaCas13a protein purified according to Kellner et al., Nature Protocols 2019,
stored as 4ul aliquots at 2mg/mL.
- LwaCas13a crRNA for detecting S gene (S-crRNA_v1:
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGCAGCACC
AGCUGUCCAACCUGAAGAAG-3’), can be ordered from Synthego
- LwaCas13a crRNA for detecting Orf1ab gene (Orf1ab-crRNA_v1:
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCCAACCUCU
UCUGUAAUUUUUAAACUAU-3’), can be ordered from Synthego
- Reporter RNA for lateral flow read out (Lateral-Flow-Reporter:
5’-/56-FAM/mArArUrGrGrCmAmArArUrGrGrCmA/3Bio/-3’), can be ordered
from IDT
For Step (3) reading out using lateral flow dipstick:
- HybriDetect Dipstick (MGHD 1), Milenia Biotec GmbH
Positive control sequences (can be produced using T7 transcription from synthesized
DNA oligos):
- S gene fragment:
5’-UAACAUCACUAGGUUUCAAACUUUACUUGCUUUACAUAGAAGUU
AUUUGACUCCUGGUGAUUCUUCUUCAGGUUGGACAGCUGGUGCUGC
AGCUUAUUAUGUGGGUUAUCUUCAACCUAGGACUUUUCUAUUAAAA
UAUAAUGAAAAUGGAACCAUUACAGAUGCUGUAGACUGUGC-3’
- Orf1ab gene fragment:
5’-UGAGUGUAAUGUGAAAACUACCGAAGUUGUAGGAGACAUUAUAC
UUAAACCAGCAAAUAAUAGUUUAAAAAUUACAGAAGAGGUUGGCCA
CACAGAUCUAAUGGCUGCUUAUGUAGACAAUUCUAGUCUUACUAUU
AAGAAACCUAAUGAAUUAUCUAG-3’
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