Materials and Methods

CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern

MATERIALS AND METHODSPlasmid DNA and virus RNA of SARS-CoV-2.SARS-CoV-2 genome sequences of different variants from different regions were downloaded from NCBI GenBank database and aligned to determine the target sequences for developing CRISPR Cas12a-based assay to detect SARS-CoV-2 mutants (Table S1 and Fig. S1 in the supplemental material). The residues are highly conserved among the same variants from different geographic locations (Fig. S1), suggesting that it is possible to find highly conserved sequences to develop a molecular test and to ensure that the molecular test could be used in clinical labs worldwide. The target sequences of SARS-CoV-2 are synthesized and subcloned into the vector pUC57 as recombinant plasmids at Sangon Biotech (Shanghai, China). The SARS-CoV-2 target sequences include (i) the wild-type (WT) gene fragment of S protein (S; nucleotides nt 21,563 to 25,384; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"MN908947","term_id":"1798172431","term_text":"MN908947"}}MN908947); (ii) the mutant gene fragments of S protein, including mutations L5F, D80A, D215G, R246I, K417N, L452R/Q, Y453F, T478K, E484Q/K, N501Y, A570D, D614G, P681H, A701V, T716I, S982A, D1118H, and P1263L; (iii) the S lineage gene fragment of open reading frame 8 (ORF8; nt 27,894 to 28,259; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"MN908947","term_id":"1798172431","term_text":"MN908947"}}MN908947); (iv) the L lineage fragment of ORF8 with mutation of S84L; and (v) the nucleoprotein (NP) gene fragment (nt 28,274 to 29,533; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"MN908947","term_id":"1798172431","term_text":"MN908947"}}MN908947). All the target sequences used for plasmid construction are available in Table S2. Recombinant plasmid DNAs were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). The plasmid copy number was calculated using the following formula: plasmid copy number (copies/μl) = 6.02 × 1023 × plasmid concentration (ng/μl) × 10−9/(plasmid length × 660).Both WT and the mutant S gene were amplified using T7 promoter-tagged primer and reverse transcribed using the HiScribe T7 high-yield RNA synthesis kit (New England Biolabs, MA, USA) and then purified using the miRNeasy serum/plasma kit (Qiagen, Hilden, Germany). In vitro-transcribed WT and mutant SARS-CoV-2 S gene RNA were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) with concentrations of 7.2 × 1011 copies/μl and 9.7 × 1011 copies/μl, respectively. The RNAs were serially 10-fold diluted to prepare a series template with 1010 to 10 copies/μl, aliquoted, and stored at −80°C until use.The SARS-CoV-2 wild-type strain (19A) isolated from a COVID-19 patient in Wuhan, China, and variants alpha (B1.1.7), beta (B.1.351), and delta (B1.617.2) isolated from imported COVID-19 patients were grown in Vero cells. Viral RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA), aliquoted, and stored at −80°C until use. Furthermore, 30-two SARS-CoV-2-positive clinical samples, including 5 wild-type strains, 11 alpha variants, 8 beta variants, and 8 delta variants were collected to evaluate the assay performance. The majority of these clinical samples were from imported COVID-19 patients from different countries and regions (Table S3). SARS-CoV-2 strains have been characterized by sequencing.Design and synthesis of crRNA.The crRNAs were designed to specifically target the mutation residues based on the alignment analysis of multiple SARS-CoV-2 genome sequences of different VOCs or VOIs from different regions (Fig. S1). We evaluated two Cas12a enzymes, i.e., LbCas12a and AsCas12a, which are expressed in Lachnospiraceae bacterium (Lb) and Acidaminococcus sp. BV3L6 (As), respectively. The mixture of Cas12a and guide RNA (crRNA) with PAM (5′-TTTN-3′, where N refers to A/G/C) specifically binds the target dsDNA to activate Cas12a to cleave the target DNA sequence. For preparation of each crRNA, DNA oligonucleotides (T7-gRNA-oligonucleotide) containing T7 promoter, conserved stem-loop sequences, and guide sequences were synthesized from Ruiboxingke Biotechnology (Beijing, China) and transcribed in vitro using the HiScribe T7 high-yield RNA synthesis kit (New England Biolabs, MA, USA) according to the manufacturer’s instructions. To purify crRNAs, the transcription reactions were treated with 4 units of DNase I (New England Biolabs, MA, USA) at 37°C for 40 min and then purified using the miRNeasy serum/plasma kit (Qiagen, Hilden, Germany). These crRNAs were measured by a NanoDrop 2000 spectrophotometer, aliquoted, and stored at −80°C until use. All the crRNA sequences used in this study are available in Table S4.Cas12a-mediated fluoresce assay.The CRISPR-Cas12a-mediated fluoresce assay for direct detection of the target has been previously described (29). In brief, 400 nM LbCas12a (New England Biolabs, MA, USA) or AsCas12a (Bio-lifesci, Guangzhou, China) was preincubated with 1,000 nM crRNA in 1× NEB buffer 2.1 at 37°C for 10 min to form crRNA-Cas12a complex followed by addition of various amounts of target DNA and 400 nM probe reporter (5′-6-FAM-TTATT-BHQ-1-3′), synthesized at Sangon Biotech (Shanghai, China), and incubated at 37°C for 1 h. Then, the fluorescence signal was monitored every 20 s on a fluorescent detector (Qitian, Jiangsu, China).To validate the assay specificity, we tested common human coronavirus (HCoV) 229E, HCoV OC43, and HCoV HKU1 as well as various other respiratory pathogens, including rhinovirus (HRV), adenovirus (ADV), respiratory syncytial virus (RSV) A and B, human bocavirus (HBoV), human metapneumovirus (HMPV), human parainfluenza virus (HPIV-1 and HPIV-4), and Mycoplasma pneumoniae. These samples were kindly provided by Jincun Zhao of State Key Laboratory of Respiratory Disease, Guangzhou Medical University (Guangzhou, Guangdong, China). Assay specificity was also validated with HIV-1 (plasmid pNL4-3; 73 ng/μl), hepatitis B virus (HBV), hepatitis C virus (HCV) (plasmid JFH; 114 ng/μl), Chlamydia trachomatis, and Treponema pallidum. The HBV DNA was extracted from the serum samples included in the national reference materials for HBV detection kit (National Institutes for Food and Drug Control, China). The C. trachomatis genome DNA was extracted from the cervical secretion of C. trachomatis-infected patients, and the T. pallidum genome DNA was extracted from the T. pallidum strain isolated from T. pallidum-infected rabbit.Combination of PCR and Cas12a-mediated fluoresce assay.Target nucleic acid sequences were first amplified by conventional PCR for DNA templates or by reverse transcription-PCR (RT-PCR) for RNA templates. The RNA template was reverse transcribed into cDNA using oligo(dT) and random primer according to the manufacturer’s instructions (Roche Diagnostics, Indianapolis, USA). To ensure the primers to account for the accumulation of diversity in the virus population, we have performed alignment analysis of multiple SARS-CoV-2 genome sequences using MAFFT version 7 (57). SARS-CoV-2 genome sequences of different variants from different regions were downloaded from the NCBI GenBank database, and the detailed information is provided in Table S1. Moreover, primers used in our assay were evaluated and validated as SARS-CoV-2 specific (target specific) by NCBI Primer-BLAST (58). The primer sequences are available in Table S5. Briefly, 12.5 μl ApexHF HS DNA polymerase FS mix (Accurate Biotechnology, Hunan, China), 1 μl of forward and reverse primers (10 μM), 8.5 μl of nuclease-free H2O, and 1 μl of target template were mixed. The reaction was run at 98°C for 30 s followed by 40 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 30 s. Finally, 1 μl of PCR product was mixed with crRNA-LbCas12a complex (1,000 nM crRNA and 400 nM LbCas12a, respectively) and 400 nM probe reporter (5′-6-FAM-TTATT-BHQ-1-3′) and incubated at 37°C for 1 h. The fluorescence was measured every 20 s on a fluorescent detector.Statistical analysis.Data were analyzed using R software version 3.5.2 (R Foundation for Statistical Computing). A two-tailed Student's t test was used to analyze the fluorescence difference between on-target and off-target templates detected by CRISPR-Cas12a-based assay. A P value of <0.05 was considered statistically significant.

Article TitleCRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern

Abstract

The crRNAs were designed to specifically target the mutation residues based on the alignment analysis of multiple SARS-CoV-2 genome sequences of different VOCs or VOIs from different regions (Fig. S1). We evaluated two Cas12a enzymes, i.e., LbCas12a and AsCas12a, which are expressed inLachnospiraceaebacterium (Lb) andAcidaminococcussp. BV3L6 (As), respectively. The mixture of Cas12a and guide RNA (crRNA) with PAM (5′-TTTN-3′, where N refers to A/G/C) specifically binds the target dsDNA to activate Cas12a to cleave the target DNA sequence. For preparation of each crRNA, DNA oligonucleotides (T7-gRNA-oligonucleotide) containing T7 promoter, conserved stem-loop sequences, and guide sequences were synthesized from Ruiboxingke Biotechnology (Beijing, China) and transcribedin vitrousing the HiScribe T7 high-yield RNA synthesis kit (New England Biolabs, MA, USA) according to the manufacturer’s instructions. To purify crRNAs, the transcription reactions were treated with 4 units of DNase I (New England Biolabs, MA, USA) at 37°C for 40 min and then purified using the miRNeasy serum/plasma kit (Qiagen, Hilden, Germany). These crRNAs were measured by a NanoDrop 2000 spectrophotometer, aliquoted, and stored at −80°C until use. All the crRNA sequences used in this study are available in Table S4.


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