Journal: bioRxiv

Article Title: A CRISPR-based SARS-CoV-2 diagnostic assay that is robust against viral evolution and RNA editing

doi: 10.1101/2020.07.03.185850

Figure Lengend Snippet: a Organization of the SARS-CoV-2 genome. ORF1a and ORF1b occupy over half of the genome. Genes encoding structural proteins are indicated by green boxes, while genes encoding accessory proteins are indicated by cyan boxes. Although ORF10 is annotated in the genome, there is currently no evidence of its expression . The locations of the new gRNAs are shown by pink bars below the genes, while the N-Mam locus is shown by a red bar. b Fluorescence measurements using a microplate reader after 30 minutes of cleavage reaction. 1E11 copies of the relevant DNA target were present in a 50μl reaction. All the readings were normalized to the negative control (NTC) at the start of the experiment. The N1 gRNA gave an unexpected result, whereby it triggered the collateral activity of AsCas12a and its variants even in the absence of a template. Data represent mean ± s.e.m. (n = 3-6 biological replicates). c Visualization of cleavage reactions for the S3 gRNA using a UV transilluminator. Transition of colours from blue to yellow to red indicates an increasing amount of collateral activity. d Sequences of perfect matched (PM) or mismatched (MM) spacers targeting the S3 locus. Each mismatched position is indicated by a bold red letter. e Collateral activity of LbCas12a complexed with perfect matched or mismatched S3 gRNA. The fluorescence measurements were taken after 30 minutes of cleavage reaction using a microplate reader and all the readings were normalized to the NTC at the start of the experiment. Our results indicate that LbCas12a should be paired with the S3 gRNA instead of the N-Mam gRNA if a diagnostic assay that is highly specific for known SARS-CoV-2 isolates is desired. Data represent mean ± s.e.m. (n = 3 biological replicates).

Article Snippet: The pET28b-T7-Cas12a-NLS-6xHis expression plasmids were gifts from Keith Joung and Benjamin Kleinstiver (Addgene plasmid #114069 [AsCas12a], #114070 [LbCas12a], #114072 [enAsCas12a], #114075 [enRVR], and #114077 [enRR]) .

Techniques: Expressing, Fluorescence, Negative Control, Activity Assay, Diagnostic Assay

Journal: bioRxiv

Article Title: A CRISPR-based SARS-CoV-2 diagnostic assay that is robust against viral evolution and RNA editing

doi: 10.1101/2020.07.03.185850

Figure Lengend Snippet: a Fluorescence measurements for a single S2 gRNA after 30 minutes of trans-cleavage reaction at 24°C. 1E11 copies of DNA template corresponding to one of the three coronaviruses (see colour bar) were present in a 50μl reaction. Spacers of three different lengths targeting the S2 locus were tested. All the measurements were normalized to the negative control (NTC) at the 0min timepoint. No cross-reactivity for SARS-CoV or MERS-CoV was detected. Unexpectedly, the 18nt spacer yielded higher fluorescence than the 19nt spacer for all the tested nucleases, especially LbCas12a. Data represent mean ± s.e.m. (n = 3-4 biological replicates). b Sequences of perfect matched (PM) or mismatched (MM) spacers targeting the S2 locus. Each mismatched position is indicated by a bold red letter. c Heatmap showing the tolerance of various Cas12a enzymes to mismatched S2 gRNAs when the trans-cleavage assay was performed at 24°C. The fluorescence readings are scaled between 0 and 1 (variable shades of green), where 1 is the highest measurement obtained at 24°C and 0 is the background signal for NTC at the start of the experiment. d Fluorescence measurements for a single S2 gRNA after 30 minutes of trans-cleavage reaction at 37°C. There was still no cross-reactivity for SARS-CoV or MERS-CoV at the higher temperature, but the fluorescence signal for SARS-CoV-2 was approximately twice as high. Data represent mean ± s.e.m. (n = 3 biological replicates). e Heatmap showing the tolerance of various Cas12a enzymes to mismatched S2 gRNAs when the trans-cleavage assay was performed at 37°C. The fluorescence readings are scaled between 0 and 1 (variable shades of red), where 1 is the highest measurement obtained at 37°C and 0 is the background signal for NTC at the start of the experiment. The most detrimental mismatch position appears to be 2nt from the PAM-distal end (MM10). f Fluorescence measurements for two PM gRNAs (S1 and S2) after 30 minutes of cleavage reaction at 37°C. No cross-reactivity for SARS-CoV or MERS-CoV was observed. Data represent mean ± s.e.m. (n = 3 biological replicates). g Heatmap showing how the addition of a second perfect matched S1 gRNA changed the tolerance of various Cas12a enzymes to mismatched S2 gRNAs. The trans-cleavage assay was performed at 37°C, with the fluorescence readings scaled between 0 and 1.

Article Snippet: The pET28b-T7-Cas12a-NLS-6xHis expression plasmids were gifts from Keith Joung and Benjamin Kleinstiver (Addgene plasmid #114069 [AsCas12a], #114070 [LbCas12a], #114072 [enAsCas12a], #114075 [enRVR], and #114077 [enRR]) .

Techniques: Fluorescence, Negative Control, Cleavage Assay