Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection.

Background: The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics.
Methods: We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability.
Results: Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions: CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19. © This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2022, corrected publication 2022.

Introduction

With a global pandemic of over 72 million COVID-19 cases resulting in over 1.6 million deaths, there remains a crucial need for diagnostic tools that allow for quick yet accurate detection of SARS-CoV-2 without the requirement of expensive equipment and extensive training[1,2]. While several vaccines are under Phase III clinical trials or have been granted Emergency Use Authorizations (EUAs) by the FDA[3-7], several variants of concern are emerging. The second, third, and fourth waves have impacted many countries across the globe, and with treatments still somewhat limited, improvements in testing are more necessary than ever to keep both case numbers and fatality numbers down in order for preventative measures to be effective down the line[8,9].

SARS-CoV-2 is a ~30 kb betacoronavirus composed of four known structural proteins, including the spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins, as well as a viral RNA genome[10]. Nucleic acid detection methods often target N, E, and RdRp (RNA-dependent RNA polymerase) genes. In quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR), the viral RNA is reverse transcribed into complementary DNA (cDNA) and then amplified through cyclic changing of temperatures to achieve denaturation of nucleic acid strands, annealing of primers to the template DNA, and extension of new complementary DNA by the addition of nucleotides by a polymerase. DNA copies are detected in real-time through the emission of fluorescence-based reporters. Detection using this method requires expensive equipment and clinically trained professionals, and is not easily portable[11,12].

CRISPR-Cas-based methods such as SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) have recently received an EUA by the Food and Drug Administration (FDA). They take advantage of the collateral cleavage (trans) activity from Class 2 Type V and Type VI Cas proteins, specifically Cas13a and Cas12a, to cleave a FRET-based reporter resulting in fluorescence. Cas12a-based DETECTR has a limit of detection of around 20 copies/µL, while SHERLOCK is based on Cas13a and is time-consuming (~1 h, as opposed to ~30 min) but can detect ~ 6.75 copies/µL (Supplementary Table S1)[13-22]. To amplify the target genes while accommodating the drawbacks of traditional PCR, both COVID-19 detection platforms pair CRISPR/Cas reactions with an antecedent isothermal amplification step, with SHERLOCK utilizing Recombinase Polymerase Amplification (RPA) and DETECTR utilizing Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP). The RT-LAMP, performed at a constant temperature of 60-65 °C, is less time-consuming due to the incorporation of loop primers that facilitate subsequent primer binding and amplification at multiple sites. It is also more sensitive than RPA and can be easily adopted without supply-chain issues[23].

Here we report the clinical validation of the ENHANCE system, which was developed in our previous study[24] and it utilizes a 7-nucleotide DNA extension on the 3’ end of the crRNA to boost the collateral cleavage activity of a LbCas12a protein, enabling an increase in sensitivity while maintaining specificity even at low magnesium concentrations. Similar to DETECTR, RT-LAMP is used for a pre-amplification step prior to the CRISPR-Cas reaction, which trans-cleaves the fluorophore from the quencher after the initial cis-cleavage of the target dsDNA. In order to validate the ENHANCEv1 system under clinical conditions, 62 nasopharyngeal samples (31 positive samples and 31 negative samples) were tested using both the fluorescence-based and lateral flow assays and then compared to RT-qPCR results. Further, we developed a lyophilized version of the ENHANCEv2 system, which utilizes modified crRNAs from ENHANCE, a mutated LbCas12a protein (Cas12aD156R) to further amplify the signal[25-28], and a dual reporter construct that allows each sample to be read using a fluorescence-based and a lateral flow assay format in the same reaction. The lyophilized CRISPR reaction in ENHANCEv2 occurs at an accelerated rate and preserves for a long period of time upon room temperature storage.

Methods

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.