COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

Abstract

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Keywords: COVID-19; CRISPR-Cas12; DETECTR; LAMP; SARS coronavirus 2; SARS-CoV-2; assay validation; guide RNAs; variant; viral mutations.

FIG 1

Design and workflow for the DETECTR assay. (A) Workflow comparison between the DETECTR assay and SARS-CoV-2 whole-genome sequencing (WGS) using either Illumina benchtop or Oxford Nanopore Technologies (ONT) nanopore sequencers. (B) Schematic of CRISPR-Cas gRNA design for SARS-CoV-2 S gene mutations. (C) Schematic of multiplexed RT-LAMP primer design showing a map of the positions of the SARS-CoV-2 S gene mutations (arrows), primers (black; black-gray), and gRNAs (blue for WT, green for MUT) within the S-gene target region. (D) Heat map comparison of three different Cas12 enzymes tested using 10 nM PCR-amplified synthetic gene fragments (t = 30 min). (E) Dot plot showing the number (n = 6) of positive RT-LAMP replicates from heat-inactivated viral cultures corresponding to known variants across a 4-log dynamic range. (F) Heat map comparison of endpoint fluorescence (t = 30 min) of three different Cas12 enzymes tested against heat-inactivated viral cultures.

FIG 2

DETECTR data analysis pipeline for SARS-CoV-2 SNP mutation calling. (A) Interpretation table summarizing the SARS-CoV-2 mutations in this study associated with corresponding lineage classifications. (B) Flow chart of the lineage classification algorithm. Scaled signals are compared across SNPs and calls are made for each RT-LAMP replicate. The combined replicate calls defines the mutation call, which informs the final lineage classification. (C, D, and E) Three representative clinical samples of different SARS-CoV-2 lineages to demonstrate the workflow of the DETECTR assay. (C) Raw fluorescence curves of each sample run in RT-LAMP amplification. (D) Subsequent triplicate DETECTR reactions targeting both WT and MUT SNPs for L452 (R), E484 (K), and N501 (Y). (E) Box plot visualization of the endpoint fluorescence in DETECTR across each SNP for the three representative clinical samples. Calls of WT, MUT, or no call were made for each SNP by evaluating the median values of the DETECTR calls corresponding to LAMP replicates. Final calls are made on the lineage determined by each SNP. Blue represents WT and green represents MUT, with RT-LAMP replicates (n = 3), CasDx1 replicates (n = 3 per LAMP replicate), and shading around kinetic curves indicates ±1.0 SD.

FIG 3

Comparison of the DETECTR assay to SARS-CoV-2 whole-genome sequencing. (A) MA plots, transformed into M (log ratio) and A (mean average) scales, show CasDx1 SNP detection replicates (n = 807) for each SARS-CoV-2 mutation across 91 clinical samples. WT is denoted by blue dots, MUT is denoted by green dots, no call is denoted by orange dots and NTC is denoted by gray dots. (B) Alignment of final mutation calls comparing the DETECTR and SARS-CoV-2 WGS assay results across 91 clinical samples after discordant samples (indicated by red asterisk) were resolved. (C) Final lineage classification on each clinical sample by the DETECTR assay compared to the SARS-CoV-2 lineage determined by viral WGS. Lineage classification categories include SARS-CoV-2 WT (blue), Alpha (red), Beta/Gamma/Mu (teal), Delta/Epsilon/Kappa (green), and Eta/Iota/Zeta (purple). (D) Final positive predictive agreement (PPA) and negative predictive agreement (NPA) values for each WT and MUT SNP from the evaluation of the DETECTR assay against the SARS-CoV-2 WGS comparator assay after discordant samples were resolved.

FIG 4

Specific detection of 484 mutations enables rapid Omicron identification. (A) Schematic of Omicron mutations within the S-gene LAMP amplicon and relative position of 484-specific gRNAs and degenerate LAMP primers. (B) Heat map comparison of endpoint fluorescence (t = 30 min) showing specific detection of 484-specific mutations (E, K, Q, A) on PCR-amplified synthetic gene fragments (n = 3). (C) SARS-CoV-2 lineage classification table based on 484 mutations. (D) Alignment of final 484 mutation calls comparing the DETECTR and SARS-CoV-2 WGS assay results across 36 clinical samples. (E) Overall SNP concordance values for the 484 SNP from the evaluation of the DETECTR assay against the SARS-CoV-2 WGS comparator assay.

FIG 5

Testing of the COVID-19 variant DETECTR assay on a validation set of 165 clinical samples. Samples are processed according to a standard clinical laboratory workflow with no biological or technical replicates. (A) Schematic showing development of DETECTR workflow. (B) Final lineage classification by the DETECTR assay, after using viral WGS and DETECTR as assay comparators. Discordant calls are denoted with arrows. (C) Table showing the number of concordant samples (major diagonal from the upper left to lower right) and discordant samples (off the major diagonal) and overall agreement for each lineage classification.