key: cord-0969539-445e6icx
authors: Fasching, C. L.; Servellita, V.; McKay, B.; Nagesh, V.; Broughton, J. P.; Brazer, N.; Wang, B.; Sotomayor-Gonzalez, A.; Reyes, K.; Streithorst, J.; Deraney, R. N.; Stanfield, E.; Hendriks, C. G.; Miller, S.; Ching, J.; Chen, J. S.; Chiu, C. Y.
title: COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme
date: 2021-11-30
journal: nan
DOI: 10.1101/2021.11.29.21267041
sha: cb5d83a6da51ea082a806a550c029dc17e98ff15
doc_id: 969539
cord_uid: 445e6icx

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants have the potential to 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 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, and N501Y mutations associated with nearly all circulating viral lineages. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all three targeted SNP mutations. We developed a data analysis pipeline for CRISPR-based SNP identification using the assay from 91 clinical samples (Ct < 30), yielding an overall SNP concordance and agreement with SARS-CoV-2 lineage classification of 100% compared to viral whole-genome sequencing. These findings highlight the potential utility of CRISPR-based mutation detection for clinical and public health diagnostics.

The emergence of new SARS-CoV-2 variants threatens to substantially prolong the 33 COVID-19 pandemic. SARS-CoV-2 variants, especially Variants of Concern (VOCs) (1, 34 2), have caused resurgent COVID-19 outbreaks in the United States (2-5) and 35

worldwide (1, 6, 7), even in populations with a high proportion of vaccinated individuals 36 (8-11). Mutations in the spike protein, which binds to the human ACE2 receptor, can 37 render the virus more infectious and/or more resistant to antibody neutralization, 38 resulting in increased transmissibility (12), and/or escape from immunity, whether 39 vaccine-mediated or naturally acquired immunity (13, 14) . Variant identification can also 40 be clinically significant, as some mutations substantially reduce the effectiveness of 41 available monoclonal antibody therapies for the disease (15). 42

Tracking the evolution and spread of SARS-CoV-2 variants in the community is critical 44 to inform public policy regarding testing and vaccination, as well as guide contact 45 tracing and containment effects during local outbreaks (16, 17) . Virus whole-genome 46 inactivated viral culture (Fig. 1F and Fig. S1B ). In comparison, LbCas12a was capable 116 of differentiating WT from MUT at position 501 on LAMP-amplified viral cultures but 117 showed much higher background for the WT target at position 452 and higher 118 background for both WT and MUT targets at position 484 for (Fig. 1F and Fig. S1B) . 119

Additionally, AsCas12a was able to differentiate WT from MUT targets at position 452 120 albeit with substantial background but was unable to differentiate WT from MUT targets 121 at positions 484 and 501 ( Fig. 1F and Fig. S1B ). From these data, we concluded that 122

CasDx1 would provide more consistent and accurate calls for the L452R, E484K and 123 N501Y mutations. We thus proceeded to further develop the assay using only the high-124 fidelity CasDx1 enzyme. 125 126 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

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The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint To develop a data analysis pipeline for calling SARS-CoV-2 SNP mutations and assign 142 lineage classifications with the DETECTR ® assay ( Fig. 2A-B) , we first used data 143 collected from SNP synthetic gene fragment controls (n=279) that included all 144 mutational combinations of 452, 484 and 501 (see Methods). Based on the control 145 sample data, we generated allele discrimination plots (34, 35) to define boundaries that 146 separate the WT and MUT signals (Fig. S4A) . Clear differentiation between WT and 147 MUT signals was observed when plotting the ratio against the average of the WT and 148

MUT transformed values on a mean average (MA) plot (34, 35) (Fig. S4B) , with 100% 149 concordance for SNP identity at positions 452, 484, and 501 for the control samples. 150 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint 151 Performance evaluation of the DETECTR ® assay using clinical samples 152

Next, we assembled a blinded dataset consisting of 93 COVID-19 positive clinical 153 samples (previously analyzed by viral WGS) and the SNP controls run in parallel. These 154 samples were extracted, amplified in triplicate RT-LAMP reactions (Fig. S2) , and 155 processed further as triplicate CasDx1 reactions for each LAMP replicate (Fig. S3) . A 156 total of nine replicates were thus generated for each sample to detect WT or MUT SNPs 157 at positions 452, 484, and 501. The DETECTR ® data analysis pipeline was then applied 158 to each sample to provide a final lineage categorization ( Fig. 2A-C) . For a biological 159 RT-LAMP replicate to be designated as either WT or MUT, the same call needed to be 160 made from all three technical CasDx1 replicates (Fig. S5A) . A final SNP mutation call 161 was made based on ≥1 of the same calls from the three biological replicates, with 162 replicates that were designated as a No Call ignored (Fig. S5A-C) . After excluding two 163 samples that were considered invalid because the fluorescence intensity from RT-LAMP 164 amplification did not reach a pre-established threshold determined using receiver-165 operator characteristic (ROC) curve analysis ( Fig. S2 and Fig. S6 ), we evaluated a total 166 of 807 CasDx1 signals from the 91 remaining clinical samples, generating up to 9 167

replicates for each clinical sample (Fig. S5B) . Differentiation of WT and MUT signals 168 according to the allele discrimination plots was more pronounced at positions 484 and 169 501 than position 452 (Fig. S4) , whereas the MA plots showed clear separation of WT 170 and MUT calls for all three positions (Fig. 3A and Fig. S4) . The variant calls made on 171 each sample were consistent with the difference in median values of the log-172 transformed signals as determined using the data analysis pipeline (Fig. S7) . 173 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint COVID-20 and COVID-63) (Fig. S8A) . Table S1 ). 218 219 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In this study, we developed a CRISPR-based DETECTR ® assay for the detection of 236 SARS-CoV-2 variants. We evaluated three CRISPR-Cas12 enzymes, two commercially 237 available (LbCas12a from NEB and AsCas12a from IDT) and one proprietary (CasDx1 238 from Mammoth Biosciences). Based on a head-to-head comparison of these enzymes, 239

we observed clear differences in performance, with CasDx1 demonstrating the highest 240 fidelity as the only enzyme able to reliably detect all three of the targeted SNPs. A data 241 analysis pipeline was developed to differentiate between WT and MUT signals with the 242 DETECTR ® assay, yielding an overall SNP concordance of 100% (272/272 total SNP 243 calls) and 100% agreement with lineage classification compared to viral WGS. Taken 244 together, these findings show robust agreement between the DETECTR ® assay and 245 viral WGS for identification of SNP mutations and variant categorization. Thus, the 246 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint DETECTR ® assay provides a faster and simpler alternative to sequencing-based 247 methods for COVID-19 variant surveillance. 248

Our results show that the choice of Cas enzyme is important to maximize the accuracy 250 of CRISPR-based diagnostic assays and may need to be tailored to the site that is 251 being targeted. As currently configured with only three SNP targets, the DETECTR ® 252 assay cannot resolve individual major variants, except for Alpha. However, given the 253 rapid emergence and dynamic shifts in the distribution of variants over time (13), it is 254 likely that tracking of key mutations, several of which are suspected to arise by 255 convergent evolution (36), rather than tracking of variants, will be critical for surveillance 256 as the pandemic continues. Furthermore, we also develop a data analysis pipeline for 257 CRISPR-based SNP calling that can readily incorporate additional targets and offers a 258 blueprint for automated interpretation of fluorescent signals that will become more 259 complex as the degree of multiplexing increases. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint study focuses on the development and validation of a variant DETECTR ® assay using 293 conventional laboratory equipment. Future work will involve implementation onto 294 automated, portable systems for use in point of care settings. 295

In the near term, we suggest the use of the DETECTR ® assay as an initial screen for 297 the presence of a rare or novel variant (e.g., carrying both L452R and E484K or carrying 298 all three SNPs) that could be reflexed to viral WGS. As the sequencing capacity for 299 most clinical and public health laboratories is limited, the DETECTR ® assay would thus 300 enable rapid identification of variants circulating in the community to support outbreak 301 investigation and public health containment efforts. Furthermore, identification of 302 specific mutations associated with neutralizing antibody evasion, such as E484K (14), 303 could inform patient care with regards to the use of monoclonal antibodies that remain 304 effective in treating the infection (15). As the virus continues to mutate and evolve, the 305 DETECTR ® assay can be readily reconfigured by validating new gRNAs and pre-306 amplification LAMP primers and gRNAs that target emerging mutations with clinical and 307 epidemiological significance. For example, we postulate that the newly emerging 308

Omicron variant, containing at least 30 mutations in the spike protein and 11 mutations 309 in the spike RGD region targeted by the assay, could be detected by increasing 310 degeneracy in the LAMP primers and adding at least one gRNA to be able to distinguish 311 this variant from the others. Over the longer term, a validated CRISPR assay that 312 combines SARS-CoV-2 detection with variant identification would be useful as a tool for 313 simultaneous COVID-19 diagnosis in individual patients and surveillance for infection 314 control and public health purposes. 315 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. product was cleaned using AMPure XP beads following manufacturers protocol at a 329 0.7x concentration. The product was eluted in nuclease-free water and normalized to 10 330 nM. All nucleic acids used in this study are summarized in Table S2 . is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint Two LAMP primer sets, each containing 6 primers, were designed to target the L452R, 361 E484K and N501Y mutations in the SARS-CoV-2 Spike (S) protein (Supplemental 362   Table) . Sets of LAMP primers were designed from a 350 bp target sequence spanning 363 the 3 mutations using Primer Explorer V5 (https://primerexplorer.jp/e/). Candidate Eleven samples were re-extracted as described above for the NP/OP swab samples 425 and evaluated by viral WGS as described above. The samples were then thawed (XXX 426 freeze/thaws) and amplified using the LAMP protocol described above and evaluated 427 using the DETECTR ® assay as described above. 428 429 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the ratio is greater than or equal to 1, then samples have amplified sufficiently. To 451 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint identify the exact score value for a qualitative QC metric, an ROC analysis was done on 452 scores and the absolute truth (Fig. S6) . 

The wildtype and mutant target guides on NTC must ideally not show any change in 467 intensity over time. The fluorescence yield for NTC must remain constant across 468

replicates, plates and close to 1. 469

On the contrary, if a sample has a fluorescence yield of 1, then it qualifies for a No Call. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint 2. If the Contrast of the sample for a SNP was between minimum and maximum 475 contrast for the plate, then the sample is assigned a NoCall. 476

3. If the Size of the sample is lower than the Size of the NTC on the plate, then the 477 sample is assigned a NoCall. 478

→ NoCall 479 were calculated without the samples that failed the LAMP reaction ( Fig. 3B and Table  505 S1). The 2x2 cross tables classify all three SNPs across all the samples between 506 sequencing and DETECTR ® technologies (Fig. 3B and Table S1 ). The data 507 transformation and statistical analysis was done in R (42). 508 509 Acknowledgments 510

We thank the UCSF Center for Advanced Technology core facility (Delsy 511 Biosciences to the extent feasible, pending scientific review and a completed material 542 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

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The copyright holder for this this version posted November 30, 2021. ; https://doi.org/10.1101/2021.11.29.21267041 doi: medRxiv preprint

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