key: cord-0776252-12bcdwgo
authors: He, Changsheng; Lin, Cailing; Mo, Guosheng; Xi, Binbin; Li, An′an; Huang, Dongchao; Wan, Yanbin; Chen, Feng; Liang, Yufeng; Zuo, Qingxia; Xu, Wanqing; Feng, Dongyan; Zhang, Guanting; Han, Liya; Ke, Changwen; Du, Hongli; Huang, Lizhen
title: Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform
date: 2021-12-02
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2021.113857
sha: bc8e229821c7f3704dd1e0f530f7a99a24589fe2
doc_id: 776252
cord_uid: 12bcdwgo

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10(−17) M (approximately 6 copies/μL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10(−16) M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

because this mutation was small deletion instead of single-base substitution (Table S2) . 123 crRNA transcription templates were prepared using 60-nt oligos containing a T7 promoter, 124 scaffold (Table S3) . After oligo-F and oligo-R annealing, the annealed products were ligated to T-125 vectors to obtain pGM-T-crRNA plasmids. Finally, transcription templates were obtained through 126 PCR amplification with Pfu DNA polymerase from pGM-T-crRNA and were purified using a Gel 127 Extraction Kit. 128

In vitro transcription of crRNAs used a HiScribe T7 High-Yield RNA Synthesis Kit. Reactions 129 were performed in 20 μL volume at 37℃ for 16 h following the manufacturer's instruction for short 130 RNA transcripts. Finally, the crRNA transcripts were treated and purified with an RNA Cleanup Kit. 131

The DNA targets of WT S gene and MT SARS-CoV-2 (covering D614G, N501Y and 69/70 133 deletion sites) were synthesized (Table S4 ) and then cloned into pUC57 plasmid. 134 RNA targets were prepared through in vitro transcription with a HiScribe T7 High-Yield RNA 135 Synthesis Kit. Briefly, RNA transcription templates containing a T7 promoter were amplified from 136 pUC57-WT-DNA and pUC57-MT-DNA with specific primers (Table S2 ) using the NEBNext Q5 137

Hot Start HiFi PCR Master Mix according to manufacturer's instruction, followed by purification 138 using a Gel Extraction Kit. In vitro transcription was performed in 20 μL reaction volume, according 139 to a standard RNA synthesis protocol, at 37℃ for 16 h. Finally, RNA transcripts were treated with Briefly, LbCas12a-mediated target cleavage assays were performed in 20 μL reaction volume 144 with 50 nM LbCas12a protein, 100 nM crRNA, 1× NEBuffer2.1, 20 U recombination RNase 145 inhibitor (RRI), 5 nM linear pUC57-WT-DNA or pUC57-MT-DNA plasmids, and nuclease-free 146 water. LbCas12a was preincubated with crRNA and RRI in NEBuffer2.1 at room temperature for 147 10 min to form ribonucleoproteins. Target DNA was then added, and the reaction mixture was 148 incubated at 37℃ for 1 or 2 h. Finally, cleaved products were verified using gel electrophoresis. 149

To investigate the identification of mutation through CRISPR/Cas12a trans-cleavage of 150 fluorescence reporter, the in vitro trans-cleavage assays were performed in 20 μL reaction volume 151 with 50 nM LbCas12a protein, 100nM crRNA, 1× NEBuffer2.1, 20 U recombination RNase 152 inhibitor (RRI), 1 nM pUC57-WT-DNA or pUC57-MT-DNA plasmids, and nuclease-free water. 153

The reaction mixtures were then quickly transferred to a 384-well fluorescence plate reader and 154 incubated at 37℃. Fluorescence signals were collected every 5 min at an excitation wavelength of 155 485 nm and an emission wavelength of 535 nm. 156

Bioinformatic alignment of multiple SARS-CoV-2 sequences downloaded from GISAID was 158 performed to identify specific and relatively conserved sequences adjacent to mutations. These 159 sequences were used to design PCR/RT-PCR primers (TableS2). 160

The modified CORDS fluorescence assay comprises two steps, PCR amplification and Cas12a 161 sensing. PCR reactions were performed in 50 μL reaction volume containing 20 μL of DNA, 4 μL 162 of 10 μM primer F/R mix, 25 μL of 2× PrimeSTAR max, and nuclease-free water. Thermal cycling 163 s at 55℃ for annealing, and 5 s at 72℃ for extension; and 5 min at 72℃ for the final extension. For 165

Cas12a reaction, FAM-and BHQ-labeled 12-nt ssDNA reporter (FQ-ssDNA) (FAM-166 NNNNNNNNNNNN-BHQ) for reporting the collateral cleavage of Cas12a was synthesized. The 167 fluorescence reporting assay was performed following a detection protocol. The whole 20 μL 168 reaction volume contained 50 nM LbCas12a, 100 nM crRNA, and 20 U RNase inhibitor, 1× 169

NEBuffer2.1, 10 μL PCR product, 500 nM FQ-ssDNA, and nuclease-free water at room temperature. 170

The reaction mixture was then quickly transferred to a 384-well fluorescence plate reader and 171 incubated at 37℃. Fluorescence signals were detected using Tecan's Spark 20M multimode 172 microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. 173

Fluorescence assay kinetics assay were assessed with signals collected every 2.5 or 5 min. 174

The PCR reaction for the CORDS lateral flow strip assay was performed as described earlier in 175 this section. Digoxin-and biotin-labeled 14-nt ssDNA reporter (DB-ssDNA) (Digoxin-176 NNNNNNNNNNNNNN-Biotin) was used to report Cas12a collateral activity in the lateral flow 177 strip reporting assay. Briefly, a Cas12a paper strip assay was performed in 40 μL reaction volume 178 containing nuclease-free water, 1× NEBuffer2.1, 40 U RNase inhibitor, 50 nM LbCas12a, 100 nM 179 crRNA, 2 nM DB-ssDNA, and 20 μL PCR product. The reaction mixture was then incubated at 37℃ 180 for 60 min. Finally, lateral flow strips were inserted into the reaction mixture and incubated at room 181 temperature for 5 min for use as readouts. 182

The RT-CORDS fluorescence assay comprises an RT-PCR reaction for amplification and a 184

Cas12a reaction for sensing. A One-Step RT-PCR Kit was used. In brief, 50μL reaction volume 185 contains 18.5 μL of RNA sample, 4 μL of 10 μM primer F/R mix, 25 μL of 2× One Step mix and 186 2.5 μL of One Step Enzyme mix. The amplification program was as follows: 30 min at 50°C for 187 reverse transcription; 3 min at 94°C for initial denaturation; 30 cycles of 30 s at 94°C for 188 denaturation, 30 s at 55°C for annealing, and 30 s at 72℃ for extension; and 5 min at 72 ℃ for the 189 final extension. The Cas12a reaction was performed using a method similar to that used for the 190 CORDS fluorescence assay, except the RT-PCR product substituted the PCR product. 191

The RT-PCR reaction for the RT-CORDS lateral strip assay was performed as described earlier 192 in this section. The Cas12a reaction was performed using a method similar to that used for the 193 CORDS lateral flow strip assay, except the RT-PCR product substituted the PCR product. 

All the replicate experiments in this study consisted of three repeats. Uncertainties in mean 202 values are provided as standard errors. Statistical analyses were performed using GraphPad Prism. 203

Statistical significance was assessed using 2-way analysis of variance and multiple comparisons. 204

Significance was considered at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns indicates 205 no significance. 206 J o u r n a l P r e -p r o o f

The principle of the biosensor was depicted in Fig. 1A . Mutant specific Cas12a/crRNA-M and 209 wild-type specific Cas12a/crRNA-W were designed to differentiate MT from WT sample. The 210 crRNAs for single-base mutation were designed by introducing a transition mismatch in the seed 211 region to enhance specificity, such as U-T mismatch in N501Y and C-C mismatch in D614G 

The SARS-CoV-2 reference genome sequence was downloaded from the National Center for 219 Biotechnology Information and mutation information for SARS-CoV-2 variants was obtained from 220 GISAID (https://www.gisaid.org/). Because CRISPR/Cas12a has been reported to differentiate 221 (Table S1 , Fig. 1A -1D) . crRNA) to detect N501Y mutation (Table S2) We have previously established a CORDS platform for in vitro virus nucleic acid detection (Bai 258 et al. 2019). For mutation identification, we also introduced fluorescence probes, 12-nt ssDNAs 259 labeled with 5′ -FAM and 3′-BHQ, to report the collateral activity of Cas12a. We assessed the 260 specificity of mutation discrimination by integrating a fluorescence reporter system. Fluorescence 261 increased solely when 501-crRNA-M was used to detect Y501-MT substrate (Fig. 1E , S1A, and 262 S1B). The same results were observed when the substrate was N501-WT, 69/70-WT, or 69/70-MT 263 (Fig. 1F, 1G , and S1C-S1F). The G614-MT substrate could only activate Cas12a/614-crRNA-M 264 but not Cas12a/614-crRNA-W. D614-MT can be slightly activated by Cas12a/614-crRNA-W (Fig.  265 1F and S1D). However, significant differences in fluorescence intensities between 614-crRNA-W 266 and 614-crRNA-M were noted, and this slight activation had no impact on D614G discrimination. 267

Furthermore, kinetic data showed that 30 min is enough time to differentiate the MT variants from 268 the WT (Fig. 1E-1G) . 269

After validating the Cas12a fluorescence assay to differentiate mutations, we introduced PCR 270 amplification to improve the sensitivity of mutation identification. The limit of detection (LOD) can 271 reach 10 -16 M when Y501-MT or N501-WT amplicons serve as substrates ( Fig. 2A, S2, S4A, and  272   S4B ). The 69/70 deletion can be identified even when substrate is present at 10 -17 M using TTC as 273 the PAM (Fig. 2B, and S3) . Thus, we developed CORDS fluorescence reporting system that rapidly 274 detects mutations from SARS-CoV-2 variants, with high sensitivity and specificity. 275 The system was made more convenient with reduced dependence of instruments when we 286 combined CORDS with lateral flow strips as a substitute for the fluorescence intensity readout (Fig.  287   2C) . In this assay, 5′-digoxin and 3′-biotin-labeled 14-nt ssDNA and lateral flow strips were used to 288 report the collateral activity of Cas12a (Fig. 2C) . Similar to the fluorescence reporting assay, we 289 detected serial concentrations of substrate to determine the sensitivity of the CORDS lateral flow 290 strips reporting system. Results indicated that LOD can reached 10 −15 M with Y501-MT or N501-291 WT as the substrate (Fig. 2D and S4C ). This sensitivity can also be achieved for 69/70-MT as the 292 substrate (Fig. 2E) . CORDS paper strips system can rapidly detect SARS-CoV-2 variants mutations, 293

with high sensitivity and without the need for a signal detection instrument. 294

The established CORDS fluorescence and paper strip systems can be used for rapid and sensitive In RT-CORDS, RT-PCR was introduced for RNA reverse transcription and amplification instead 319 of isothermal amplification because the latter is always unstable when combined with reverse 320 transcription (Fig.S5) . LOD reached 10 −17 M with Y501-MT RNA as substrate (Fig. 3A, 3B) . The 321 sensitivity of the RT-CORDS fluorescence reporting system was higher than that of the CORDS 322 system, perhaps because of the different amplification ability of the DNA polymerase. The 323 sensitivity of RT-CORDS for N501Y detection was 10 −15 M, the same sensitivity as that of CORDS, 324 when lateral flow strips were used as final read out (Fig. 2D, 4A, and 4B ). RT-CORDS fluorescence 325 assay for 69/70 deletion identification showed a sensitivity of 10 −17 M, the same value as that 326 achieved for N501Y ( Fig. 3C and 3D) . The LOD for RT-CORDS lateral flow strip was 10 −16 M (Fig.  327 4C and 4D), higher than observed for N501Y detection and in a previous study (Bai et al. 2019) . 328

The sensitivity for D614G detection was 10 −17 M for RT-CORDS fluorescence and 10 −15 M for RT-329 CORDS paper strip reporting systems (Fig. 3E, 3F, 4E, and 4F) . Increased fluorescence and test (Table S5) . Notably, 334 kinetic analyses showed N501Y and 69/70 deletion can be identified in as small duration as 5-10 335 min, indicating a shorter reaction time for mutation tracking (Fig. S6 and S7) . However, 40 min is 336 the most advisable for D614G detection (Fig. S8) . 

In total, 18 SARS-CoV-2 variant RNA samples were tested using the RT-CORDS paper strip 345 system. All samples carried the D614G mutation (Fig. 5A) . Furthermore, five samples carrying the 346 N501Y mutation and two samples carrying the 69/70 deletion were identified (Fig. 5B-5D) . Results 347 were 100% consistent with the sequencing data ( Fig. 5 and S9 -S11). 348

We simplified the RT-CORDS procedure for storage and use through the lyophilized paper 349 strip system (Fig. S12A) . Lyophilization did not alter the LOD for mutation identification (Fig.  350   S12B) . Besides, according to our accelerated stability test, the lyophilization of Cas12a mix work 351 well after storage at 4 ℃ for 1 week without loss of sensitivity (Fig. S12C) . RT-CORDS is a rapid, 352 robust, and accurate method for the identification of SARS-CoV-2 N501Y, D614G, and 69/70 353 deletion mutations and may be used for additional tracking of spike mutations through a simple 354 process in clinical diagnostics ( Fig. 6 and S13) . indicated that all the designed specific crRNAs worked well for different types of mutation, yet 371 without laborious screening process (Fig. 1B-G) . 372

High accuracy is the key critical benefit of the RT-CORDS. By introducing a skillful point 373 mutation at the seed region of crRNA, the RT-CORDS can detect the SARS-CoV-2 single-base 374 mutations (N501Y and D614G) with high specificity. RT-CORDS can also accurately identify a 375 small deletion mutation in SARS-CoV-2 (69/70 deletion) with specific crRNAs without introducing 376 a point mutation. In this study, 18 SARS-CoV-2 variants were detected, and all three mutations 377 identified by RT-CORDS were perfectly consistent with the sequencing data, indicating 100% 378 accuracy (Fig. 5) . Compared with similar CRISPR-based methods, the accuracy in this study was 379 higher than that of a Cas9-based N501Y detection method (accuracy: 86%) (Kumar et al. 2021) . The RT-CORDS differentiates mutations in SARS-CoV-2 variants accurately and ehibit high 392 sensitivity for all three SARS-CoV-2 mutation targets. RT-CORDS with fluorescence showed LODs 393 for all three mutations reached 10 -17 M (approximately 6 copies/μL) (Fig. 3, Table S5 ), which was 394 higher than that of the Cas13-based N501Y detection system (100 copies/μL) (de Puig et al. 2021). CORDS is, therefore, sufficiently sensitive to facilitate the early screening of SARS-CoV-2 variants. 407

The narrow range of targets makes the PAM sequences a key obstacle for designing an efficient 408 crRNA for mutation detection. We used TTC as an alternative PAM for 69/70 deletion. No TTTV 409 sequence was present near the 69/70 site. Although several studies have reported a lower affinity of 410 TTV for Cas12a loading, the LOD for the 69/70 deletion was the same as that for canonical PAM, 411

indicating that TTV can also be efficient for activating Cas12a system sensitively (Fig. 2B, 3C , and 412 4C) (Yamano et al. 2017) . Sequences adjacent to the S gene mutations of SARS-CoV-2 VOC and 413 VOI revealed TTTV and TTV near all mutations, indicating that these mutations are targetable and 414

can be tracked using RT-CORDS (Fig. 6) . Conversely, RAY, a Cas9-based SARS-CoV-2 variant in mutation detection as optimal primer design is limited within the narrow range of mutant targets. 426

The new SARS-CoV-2 strain shows a stronger binding affinity 458 to ACE2 due to N501Y mutant

Development of a genotyping platform for SARS

CoV-2 variants using high-resolution melting analysis

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine 464

Two-step 468 strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants 469 with spike deletion H69-V70

Implementation of an in-house real-time reverse transcription-474 PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant

Limitation of Screening of

dsmCRISPR: Dual synthetic mismatches

CRISPR/Cas12a-based detection of SARS-CoV-2 D614G mutation

Massively parallel kinetic profiling of natural and 542 engineered CRISPR nucleases

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G

Increases Infectivity of the COVID-19 Virus

FnCas9 based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-552 2 variants on a paper strip

Experimental Evidence for 554

Enhanced Receptor Binding by Rapidly Spreading SARS-CoV-2 Variants

CRISPR Diagnosis and Therapeutics 557 with Single Base Pair Precision

Detection of rare variant alleles using the AsCas12a 559 double-stranded DNA trans-cleavage activity

An ultrasensitive CRISPR/Cas12a based electrochemical 562 biosensor for Listeria monocytogenes detection

SARS-CoV-2 501Y

Recurrent emergence of SARS-CoV-2 spike 584 deletion H69/V70 and its role in the Alpha variant B.1.1.7

Detection of the SARS-CoV-2 D614G mutation using 587 engineered Cas12a guide RNA

Viral load of SARS-CoV-2 in clinical 589 samples

Spike mutation D614G alters SARS-CoV-2 fitness

Aptamer-locker" DNA coupling 595 with CRISPR/Cas12a-guided biosensing for high-efficiency melamine analysis

CRISPR Enzyme Kinetics for Molecular Diagnostics

SARS-CoV-2 SPIKE PROTEIN: an optimal immunological target for vaccines

The emerging SARS-CoV-2 variants of concern. Therapeutic Advances in Infectious 604

The effect of 606 primer-template mismatches on the detection and quantification of nucleic acids using the 5' 607 nuclease assay

Studying the effect of single mismatches in primer 609 and probe binding regions on amplification curves and quantification in real-time PCR

Structural Basis 626 for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

Variant of Concern by Combining Novel PCR Assays and Genome Sequencing for Rapid

CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

Bioelectronics 169, 112642. 495Daniloski, Z., Jordan, T.X., Ilmain, J.K., Guo, X., Bhabha, G., tenOever, B.R., Sanjana, N.E., 2021. 496The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types. 497Elife 10, e65365.