FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip
- CSIR-Institute of Genomics & Integrative Biology, India
- Academy of Scientific & Innovative Research (AcSIR), India
- CSIR-National Chemical Laboratory, India
Figures
Figure 1
Schematic for RAY.
(A) FnCas9 is unable to bind or cleave targets having two mismatches at the PAM proximal 2nd and 6th or PAM distal 16th and 19th positions as shown (left panel). The quantification of cleavage with …
Figure 2
Adaptation of RAY for identification of N501Y mutation.
(A) Schematic showing the application of RAY for distinguishing variants WT and N501Y using electrophoresis, key steps in the process are shown. The uncleaved band represents the amplicon from …
Figure 3
Validation of single amplicon RAY on patient samples.
(A) RAY optimization with different size of S gene PCR amplicons. Optimal amplicon length denoted by red dotted box. (B) Reproducibility in output on multiple runs of RAY on the same samples (WT or …
Figure 4
Double amplicon RAY.
(A) Effect of single/double biotin labeling on primers for the 580 bp amplicon. (B) Effect of reduction of amplicon length on RAY outcomes. Red dotted box shows the optimal amplification conditions …
Appendix 1—figure 1
Cycling conditions for Reverse Transcription-PCR for RAY .
Appendix 1—figure 2
Representative image of RAY outcomes on a paper strip for sample having the N501Y mutation (Mut) or the parent CoV-2 sequence (Wt).
Left panel shows outcome with SN501Y sgRNA and the right panel shows the outcome with SWT sgRNA.
Appendix 1—figure 3
Representative screen-shots showing TOPSE app home page and user interface.
Appendix 1—figure 4
Representative screen-shots showing TOPSE image acquisition for NTC background correction.
Appendix 1—figure 5
Representative screen-shots showing TOPSE data entry step and image acquisition for a sample identified as ‘’Positive’’.
Appendix 1—figure 6
Representative screen-shots showing TOPSE data entry step and image acquisition for a sample identified as “Negative’’.
Additional files
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Supplementary file 1
Mutations across emerging SARS-CoV-2 lineages showing the possibility of RAY based targetability of VOCs/VOIs.
- https://cdn.elifesciences.org/articles/67130/elife-67130-supp1-v2.xlsx
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Supplementary file 2
RAY outcomes on patient samples (WT or N501Y).
The cutoff for SWT is 6.7 and SN501Y is 10.2.
- https://cdn.elifesciences.org/articles/67130/elife-67130-supp2-v2.xlsx
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Supplementary file 3
Price estimate of RAY consumables.
- https://cdn.elifesciences.org/articles/67130/elife-67130-supp3-v2.xlsx
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Supplementary file 4
List of oligos used in this study.
- https://cdn.elifesciences.org/articles/67130/elife-67130-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67130/elife-67130-transrepform-v2.pdf
