Protein expression and purification
LbCas12a gene fragment was obtained by PCR from the plasmid LbCpf1-2NLS as a gift from Jennifer Doudna (Addgene plasmid # 102566)29 and subcloned into a linearized CL7-tagged vector (Plasmid #21, TriAltus Bioscience) by digesting with HindIII and XhoI. The fragments assembly was performed using NEBHiFi Builder (New England Biolabs) following the manufacturer’s protocol. The assembled plasmid was transformed into DH5ɑ (New England Biolabs) competent cells. Individual colonies were picked the next day and inoculated in 10 ml of LB broth, Miller (Fisher Scientific) at 37 oC overnight. Cells were harvested, and plasmids were extracted and purified using the Monarch Mini Plasmid prep (New England Biolabs). The CL7-tagged LbCas12a and LbCas12aD156R expression plasmids are made available on Addgene (#164658 and 164659, respectively).
For protein expression, 100 ng of the purified plasmid was transformed into BL21(DE3) competent cells (New England Biolabs). Individual colonies were picked and inoculated in 10 ml of Terrific Broth (TB) at 37 oC for 8–10 h. The culture was then added to 1 L TB broth containing 50 µg/ml Kanamycin (Fisher Scientific) and 50 µL antifoam 204 (Sigma Aldrich) and let grown until OD 600 = 0.6. The culture was then taken out of the 37 oC incubator and let cooled on ice for 30–45 min. Next, 0.5 mL of 1M isopropyl β-d-1-thiogalactopyranoside (IPTG, Fisher Scientific) was added to the culture and let grown at 18 oC overnight.
The overnight culture was centrifuged to collect cell pellets the next day. They were resuspended in Lysis Buffer A (2 M NaCl, 50 mM Tris-HCl, pH = 7.5, 0.5 mM TCEP, 5% Glycerol, 1 mM PMSF, 0.25 mg/ml lysozyme). The mixture was disrupted by sonication, centrifuged at 40,000×g, and filtered through a 0.45 µm filter. The lysate was run through a 1 ml CL7/Im7 column (TriAltus Bioscience) connected to the FPLC Biologic Duoflow system (Bio-Rad). The column was washed for at least three cycles of alternating Wash Buffer B (2 M NaCl, 50 mM Tris-HCl, pH = 7.5, 0.5 mM TCEP, 5% Glycerol) and Wash Buffer C (50 mM Tris-HCl, pH = 7.5, 0.5 mM TCEP, 5% Glycerol). The column was then eluted by adding 5 mL of SUMO protease (purified from plasmid pCDB302 as a gift from Christopher Bahl, Addgene plasmid# 113673)30 and flown through in a closed-loop cycle at 30 oC for 1.5 h. Optionally, to remove SUMO protease, the eluted solution was then concentrated using a 30 kDa MWCO Sartorius Vivaspin Concentrator to 500 µL and subjected to size exclusion chromatography in SEC buffer (500 mM NaCl, 50 mM Tris-HCl, pH = 7.5, 0.5 mM TCEP) via the Superdex 200 increase 10/300 GL column (Cytiva). Eluted fractions were collected, pooled together, concentrated, quantified using the NanoDrop (Thermo Fisher), snap-frozen in dry ice, and stored at −80 oC until use.
For LbCas12aD156R expression and purification, the CL7-tagged plasmid obtained by subcloning above was mutated using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s protocol. The protein expression and purification were the same as described above.
Bst-LF polymerase expression plasmid was obtained as a gift from Drew Endy & Philippa Marrack (Addgene plasmid # 153313). Br512 (an engineered version of Bst polymerase) and reverse transcriptase RTx (exo-) were obtained as a gift from Andrew Ellington (Addgene plasmid # 161875 and # 145028, respectively). Bst-LF polymerase and Br512 polymerase were expressed and purified following Maranhao et al.31, and RTx(exo-) was expressed and purified following Bhadra et al.32.
Lyophilization of ENHANCEv2 CRISPR reaction
To assemble a CRISPR reaction, 100 nM LbCas12aD156R, 125 nM crRNA-Mod, 500 nM dual reporter were combined in 1x NEBuffer 2.1 (New England Biolabs) to a total of 50 µL. The mixture was scaled up accordingly to make 5x and 20x reaction aliquots. These aliquots were then subjected to lyophilization using the Labconco freeze dryer for 2–4 days.
Lyophilization of RT-LAMP reagents
One reaction of the RT-LAMP assay reagent mixture was prepared by combining 35 nanomoles dNTPs, 2.5 µL of 10X LAMP primer mix, 25 picomoles of Br512 (or Bst-LF) polymerase, 0.1 µg of RTx(exo-), and 1.25 µmoles of d-( + )-trehalose, anhydrous. The mixture was frozen for 2 h at −80 oC prior to freeze-drying using the Labconco lyophilizer for 24 h.
The lyophilized reaction mixture was reconstituted with 1X isothermal buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.4 M Betaine, PH = 8.8 at 25 oC). The RT-LAMP reaction was then readily initiated by adding RNA samples.
CDC RT-qPCR assay
The samples were re-validated with real-time RT-qPCR. The reactions were performed using the CDC-recommended Quantabio qScript XLT One-Step RT-qPCR ToughMix (Catalog# 95132-100) and TaqMan probes and primer sets and measured using the ViiA 7 Real-Time PCR System. RT-qPCR quantification was performed using amplification plots generated by the ViiA 7 software.
Viral nucleic acid extraction
For crRNA screening and optimization, LoD estimation, inclusivity testing, and specificity testing, viral RNA extraction was performed using the Lucigen QuickExtract™ DNA Extraction Solution (Cat # QE09050). Viral samples were diluted with QuickExtract™ in a 1:1 (v/v) ratio and incubated at 65 °C for 15 min and 98 °C for 2 min.
For clinical validation experiments, all patient samples were extracted using Maxwell® RSC 16 automated nucleic acid extraction instrument. Maxwell® RSC Viral Total Nucleic Acid Purification Kit (Cat# AS1330, as recommended by CDC) was used for all extractions following the manufacturer’s protocol.
RT-LAMP reactions
A set of six LAMP primers were designed for each gene using the freely available PrimerExplorer software (https://primerexplorer.jp/e/)33. The designed primers were synthesized by Integrated DNA Technologies. A 10x primer mix for each gene was created by mixing the six primers to a final concentration of 16 µM (FIP/BIP), 8 µM (LB/LF), and 2 µM (F3/B3). RT-LAMP master mix (including the positive and negative control) were prepared by combining the WarmStart® Colorimetric LAMP 2X Master Mix with UDG (New England Biolabs) and 10X LAMP primer mixes to a 1X final concentration and total volume of 20 µL. The RT-LAMP reaction was initiated by the addition of target RNA and incubated at 65 °C for 30 min to allow for adequate amplification. The amplified products were tested downstream using the CRISPR-ENHANCE assays.
Screening of crRNAs and optimization of ENHANCE for detection of SARS-CoV-2
Genomic RNA from SARS-CoV-2, Isolate USA-WA1/2020 (NR-52285) obtained from Biodefense and Emerging Infections Research Resources Repository (BEI resources), was spiked in the nucleic acid extract obtained from a healthy donor. The spiked extract was amplified for the target genes (N1, N2, E1, E2, R1, and R2) using RT-LAMP. The amplified products were then detected using wild-type CRISPR/Cas12a as well as CRISPR ENHANCEv1.
Inclusivity testing
SARS-CoV-2 Genomic RNA of isolates obtained from different geographic regions such as Italy, Hong Kong, and the USA (NR-52498, NR-52388, and NR-52285) obtained from BEI resources were spiked in the nucleic acid extract obtained from the nasopharyngeal swab of a healthy donor. Target genes were amplified using the RT-LAMP protocol described above and detected using CRISPR ENHANCEv1.
Specificity testing
To demonstrate the specificity of our assay towards SARS-CoV-2 we obtained 31 highly similar and commonly circulating pathogens from ZeptoMetrix (Cat# NATPPQ-BIO, Cat# NATRVP-3, Cat# NATPPA-BIO). Each pathogen was spiked in a matrix composed of a nasopharyngeal swab from a healthy donor. The spiked nasal swab was extracted using Lucigen QuickExtract™ and target genes within the extracted nucleic acids were amplified using RT-LAMP. The amplified products were detected using CRISPR ENHANCEv1.
Estimation of LoD
A nasopharyngeal swab sample from a healthy donor was mixed with an equal volume of Lucigen QuickExtract™ DNA Extraction Solution (Cat # QE09050) and incubated at 65 °C for 15 min and 98 °C for 2 min to extract nucleic acids from the swab. Mock clinical patient samples were prepared by serially diluting the nucleic acid extract with Quantitative PCR (qPCR) Control RNA from Heat-Inactivated SARS-Related Coronavirus 2 (BEI NR-52347) to a final concentration range of 200 copies/µL to 0.2 copies/µL. The LoD for each gene was determined by amplifying the gene using RT-LAMP and then detecting it at the indicated concentrations with CRISPR ENHANCEv1. The LoDs for the N2 gene and E2 gene were confirmed by testing with 20 replicates at 1x and 2x of the previously estimated LoD for each gene.
Fluorescence-based reporter detection assay
All fluorescence-based detection experiments were performed in a 384-well plate. CRISPR/Cas complexation was carried out by combining 30 nM LbCas12a and 60 nM crRNA in 1X NEBuffer 2.1 and incubating at 37 °C for 15 min before transferring to a 384-well plate containing 500 nM Fluorophore-Quencher (FQ) and 2 µL of RT-LAMP product. Emitted fluorescence resulting from Cas12a-based trans-cleavage was measured using BioTek Synergy 2 microplate reader with fluorescence measurement at excitation and emission wavelengths of 485/20 and 528/20, respectively, every 2.5 min. For a 96-well plate format, all reagents are scaled up 2.5 times.
Lateral flow detection assay
The detection reaction for lateral flow assay was carried out by combining 30 nM LbCas12a, 60 nM crRNA, 200 nM FAM-Biotin reporter in 1X NEBuffer 2.1 to a total volume of 48 µL. Two microliters of the corresponding RT-LAMP product was added to the above mixture and incubated at 37 °C for 20 min. A HybriDetect 1 lateral flow strip (Milenia Biotech) was then dipped in the reaction tube and the presence or absence of the target gene was determined based on a visual readout after 2 min. Lateral flow band signals were later quantified by ImageJ.
Clinical validation of patient samples using CRISPR-ENHANCE
A pool of 62 patient samples underwent a blind test. Random samples were selected from the pool, and nucleic acids extracted from those patient samples were subjected to RT-LAMP-based amplification of the N2 gene, E2 gene, and RNASE P gene. The same amplified RT-LAMP products were then detected using both the fluorescence-based reporter detection assay and the lateral flow assay with ENHANCEv1 and with the lyophilized ENHANCEv2.
Ethical statement
This study was performed under the University of Florida (UF) Institutional Review Board (IRB) protocol IRB202000781, which was approved as a non-human study, and all relevant ethical regulations were followed. De-identified human samples were obtained from the UF Clinical and Translational Science Institute (CTSI) Biorepository, collected under the UF IRB approved protocol IRB20200879, and from a commercial vendor, Boca Biolistics, procured under the IIRB delinking protocol SOP 10-00114 Rev E.
The CTSI Biorepository was approved to collect specimens without informed consent due to the COVID-19 pandemic being an unprecedented public health emergency and it would limit the research if all samples were not included. There was also the option of obtaining informed consent wherever possible. There were specific limits in the amount and type of data allowed to be gathered for those samples collected without informed consent. Some samples being tested for COVID-19 by the UF Pathology Lab came from patients in outlying clinics or hospitals. Although PHI was collected with the samples, no identifiable data or tissue was nor will be subsequently dispensed. All connections of tissue with data have been and will be conducted by honest brokers.
Boca Biolistics (BBL) is an FDA-recommended provider of SARS-CoV-2 biospecimens for research and diagnostic development. BBL provides remnant SARS-CoV-2 swab specimens as remnant (leftover) samples procured from their network of CAP/CLIA accredited partner laboratories across the United States all of whom have been instrumental in providing COVID-19 screening throughout the pandemic. Under Boca’s IIRB Delinking protocol samples are procured and de-linked so that no information can be traced back to the individual patient, providing sound and secure de-identification protecting patient identity. Boca’s SOP is consistent with the FDA’s “Guidance on informed consent for in vitro diagnostic device studies using leftover human specimens that are not individually identifiable”. This allows BBL to provide tens of thousands of highly needed SARS-CoV-2 swab specimens that have been instrumental in both the development and validation of diagnostic instruments throughout the world to test for COVID-19.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
