TITLE: A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR
AUTHORS: Mammoth Biosciences, James P. Broughton, Wayne Deng, Clare L. Fasching, Jasmeet Singh, Charles Y. Chiu, Janice S. Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">***DISCLAIMER: This protocol has not been approved by the FDA and should not be used as a clinical diagnostic***</div><div class = "text-block"><span style = "font-weight:bold;">Introduction</span></div><div class = "text-block">Given the global health emergency, rapid transmission, and severe respiratory disease associated with the outbreak of the 2019 novel coronavirus (SARS-CoV-2), Mammoth Biosciences has reconfigured our DETECTR platform to rapidly and accurately detect SARS-CoV-2 using a visual lateral flow strip format within 30 minutes from sample to result. To ensure specificity of detection, we selected a high-fidelity CRISPR detection enzyme and designed sets of gRNAs that can either 1) differentiate SARS-CoV-2 or 2) provide multi-coronavirus strain detection. SARS-CoV-2 DETECTR couples CRISPR detection with isothermal pre-amplification using primers based on protocols validated by the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). Currently in the United States, the CDC SARS-CoV-2 real-time RT-PCR diagnostic panel has a laboratory turnaround time of approximately 4-6 hours, with results that can be delayed for >24 hours after sample collection due to shipping requirements. In addition, these tests are only available in CDC-designated public health laboratories certified to perform high-complexity testing. </div><div class = "text-block">Mammoth is working to enable point of care testing (POCT) solutions that can be deployed in areas at greatest risk of transmitting SARS-CoV-2 infection, including airports, emergency departments, and local community hospitals, particularly in low-resource countries. Leveraging an “off-the-shelf” strategy to enable practical solutions within a short time frame, we describe here a protocol that is fast (<30 min), practical (available immediately from international suppliers), and validated using contrived samples.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/dde1dab5aa27f40ede8b8aab229b3a12e11b4ee97a2e4206a60448c04cd10985/b6uibb2vf.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/dde1dab5aa27f40ede8b8aab229b3a12e11b4ee97a2e4206a60448c04cd10985/b6ujbb2vf.png" /></div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "1" colspan = "2" style ="display : table-cell;text-align:center;">Specifications</td><td rowspan = "" colspan = "" style ="display : none;text-align:center;"></td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;font-style:italic;">Targets</td><td rowspan = "" colspan = "" style ="display : table-cell;">● N-gene (SARS-CoV-2 specific)
● E-gene (SARS-CoV, bat-SARS-like-CoV, and
SARS-CoV-2 coronaviruses)
● RNase P (human sample control)</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;font-style:italic;">Limit of detection</td><td rowspan = "" colspan = "" style ="display : table-cell;">10 copies/μl input</td></tr></table><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/dde1dab5aa27f40ede8b8aab229b3a12e11b4ee97a2e4206a60448c04cd10985/b6utbb2vf.png" /></div><div class = "text-block"><span style = "font-weight:bold;"> </span><span style = "font-weight:bold;">Figure 1 | a) </span><span>SARS-CoV-2 DETECTR has a limit of detection (n=6) of 10 copies per µl input. </span><span style = "font-weight:bold;">b)</span><span> Schematic of DETECTR assay coupled to lateral flow strip. </span><span style = "font-weight:bold;">c)</span><span> Representative lateral flow results for the assay shown for 0 copies per µl and 10 copies per µl</span></div><div class = "text-block"><span style = "font-style:italic;">Acknowledgements:</span><span> We thank Vikram Joshi, Nefeli Tsaloglou and Xin Miao for advice and helpful discussions in the preparation of this whitepaper.</span></div><div class = "text-block"><span style = "font-style:italic;">Conflicts of Interest:</span><span> JPB, CLF, JS and JSC are employees of Mammoth Biosciences, CYC is on the Scientific Advisory Board of Mammoth Biosciences, and JSC is a co-founder of Mammoth Biosciences. JPB, CLF, JS, CYC and JSC are co-inventors on CRISPR-related technologies.</span></div></div>

[STEPS]
?. [Prepare nucleic acid sample and CRISPR reagents]
Extract patient RNA following CDC recommendations.
?. [Prepare nucleic acid sample and CRISPR reagents]
Prepare LbCas12a RNP complexes for the samples to be tested. One complex for N-gene, E-gene, and RNase P gRNAs is needed for each sample.  ABC1ReagentVolumeFinal Concentration2Nuclease-free water15.75 μl310X NEBuffer 2.12 μl1X41 μM LbCas12a1 μl50 nM51 μM gRNA1.25 μl62.5 nM6TOTAL VOLUME20 μl
ABC1ReagentVolumeFinal Concentration2Nuclease-free water15.75 μl310X NEBuffer 2.12 μl1X41 μM LbCas12a1 μl50 nM51 μM gRNA1.25 μl62.5 nM6TOTAL VOLUME20 μl
?. [Prepare nucleic acid sample and CRISPR reagents]
Incubate LbCas12a with gRNA to generate RNP complexes for  at .
37 °C
?. [Prepare nucleic acid sample and CRISPR reagents]
Add reporter substrate to final concentration of .
?. [Prepare nucleic acid sample and CRISPR reagents]
Place reactions  until ready to proceed.
on ice
Complexes are stable at 4°C for at least 24 hours.
?. [Run DETECTR reaction]
, prepare three RT-LAMP reactions, one each for N-gene, E-gene, andRNase P primer sets:  ABC1ReagentVolumeFinal Concentration210X Isothermal Amplification Buffer (NEB)2.5 μl3100 mM MgSO 4 (NEB)1.13 μl6.5 mM (4.5 mM added, 2 mM in 1X IsoAmp Buffer)410 mM dNTPs (NEB)3.5 μl1.4 mM510X Primer Mix2.5 μl0.2 μM F3 / 0.2 μM B3 / 1.6 μM FIP / 1.6 μM BIP / 0.8 μM LF / 0.8 μM LB6Bst 2.0 polymerase (NEB)1 μl8 units / rxn7Warmstart RTx (NEB)0.5 μl7.5 units / rxn8Nuclease-free water3.87 μl9Nucleic acid sample5 μl10TOTAL VOLUME25 μl
on ice
ABC1ReagentVolumeFinal Concentration210X Isothermal Amplification Buffer (NEB)2.5 μl3100 mM MgSO 4 (NEB)1.13 μl6.5 mM (4.5 mM added, 2 mM in 1X IsoAmp Buffer)410 mM dNTPs (NEB)3.5 μl1.4 mM510X Primer Mix2.5 μl0.2 μM F3 / 0.2 μM B3 / 1.6 μM FIP / 1.6 μM BIP / 0.8 μM LF / 0.8 μM LB6Bst 2.0 polymerase (NEB)1 μl8 units / rxn7Warmstart RTx (NEB)0.5 μl7.5 units / rxn8Nuclease-free water3.87 μl9Nucleic acid sample5 μl10TOTAL VOLUME25 μl
?. [Run DETECTR reaction]
Incubate at  for .
62 °C
Note: Use precaution when opening amplification tubes to preventamplicon contamination.
?. [Run DETECTR reaction]
Combine  of the RT-LAMP reaction with  of the LbCas12a RNPcomplex with the appropriate gRNA.
2 µl
18 µl
?. [Run DETECTR reaction]
Add .
[1X NEBuffer 2.1]
?. [Run DETECTR reaction]
Incubate at  for .
37 °C
?. [Run DETECTR reaction]
Insert Milenia HybriDetect 1 (TwistDx) lateral flow strip directly into reaction.
?. [Run DETECTR reaction]
Allow the lateral flow strip to run for  at  and observethe result.
37 Room temperature
?. [Test interpretation]
ABCD1N-geneE-geneRNase PInterpretation2+++/-SARS-CoV-2 positive3+-+/-Indeterminate4-++/-Indeterminate5--+SARS-CoV-2 negative6---QC failure
Note: The line closest to the sample pad is the control line and the line that appearsfarthest from the sample pad is the test line (see Figure 1). A sample with completecleavage of the reporter molecule may appear to have no signal at the control line.
ABCD1N-geneE-geneRNase PInterpretation2+++/-SARS-CoV-2 positive3+-+/-Indeterminate4-++/-Indeterminate5--+SARS-CoV-2 negative6---QC failure