Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants

Version published to 10.1038/s41551-022-00889-z

May 30, 2022

SciScore for 10.1101/2021.11.01.21265764: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
At the Integrated Research Facility (IRF) - Frederick, the virus was passaged by inoculating grivet kidney epithelial Vero cells (ATCC #CCL-81) at a multiplicity of infection (MOI) of 0.01 under high containment (BSL-3) conditions.	Vero suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
The resulting viral seedstock was harvested and quantified by plaque assay using Vero E6 cells (ATCC #CRL-1586) with a 2.5% Avicel overlay and stained after 48 hours with a 0.2% crystal …

SciScore for 10.1101/2021.11.01.21265764: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
At the Integrated Research Facility (IRF) - Frederick, the virus was passaged by inoculating grivet kidney epithelial Vero cells (ATCC #CCL-81) at a multiplicity of infection (MOI) of 0.01 under high containment (BSL-3) conditions.	Vero suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
The resulting viral seedstock was harvested and quantified by plaque assay using Vero E6 cells (ATCC #CRL-1586) with a 2.5% Avicel overlay and stained after 48 hours with a 0.2% crystal violet stain.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
ADAPT was then run on the S gene alignment with the following parameters: 35-65% GC content, max.	ADAPT suggested: (ADAPT, RRID:SCR_006769)
Infected cells were incubated for 48 or 72 hours in Dulbecco’s Modified Eagle Medium with 4.5g/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate (DMEM, Gibco) containing 2% heat-inactivated fetal bovine serum (SAFC Biosciences) in a humidified atmosphere at 37°C with 5% CO2.	SAFC suggested: (SAFC, RRID:SCR_008554)
Single-step SARS-CoV-2 SHINE reactions: RPA primer and crRNA optimizations were performed using the following SHINE conditions: 1X original SHINE buffer (20 mM HEPES pH 8.0 with 60 mM KCl and 3.5% PEG-8000), 45 nM LwaCas13a protein resuspended in 1X SB (such that the resuspended protein is at 2.26 µM), 125 nM polyU quenched FAM reporter, 2 mM of each rNTP, 1 U/µL murine RNase inhibitor, 1 U/µL NextGen T7 RNA polymerase, 0.1 U/µL RNase H (NEB), 2 U/µL Invitrogen SuperScript IV (SSIV) reverse transcriptase (Thermo Fisher Scientific), an assay specific concentration of forward and reverse RPA primers (detailed below), and 22.5 nM crRNA.	NextGen suggested: None
BinaxNow COVID - 19 Antigen self-test - clinical samples: BinaxNow COVID-19 Antigen self-tests (Abbott) were purchased from Walmart.	Abbott suggested: (Abbott, RRID:SCR_010477)
Data panels were primarily generated using Prism 8 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

SHINEv2 addresses these limitations. Lyophilization considerably simplifies the assay and facilitates its transportation and storage. SHINEv2 can be distributed overseas without a loss in performance. Moreover, the use of an equipment-free and ambient-temperature sample lysis method further increases the user-friendliness of the assay. SHINEv2 involves as few steps from the user as antigen-capture tests, while providing a 50-fold boost in sensitivity7,42,43. Importantly, SHINEv2 demonstrates perfect (100%) concordance with RT-qPCR, the gold standard for SARS-CoV-2 diagnosis, in samples with RNA levels above our analytical LoD of 200 copies/μL. This level of sensitivity could enable the detection of every potentially infectious individual, including those missed by antigen-capture tests10,46. SHINEv2 can accurately identify several clade-specific mutations in the Alpha, Beta, Gamma and Delta SARS-CoV-2 VOCs, and it can be rapidly adapted to respond to emerging viral variants as well as other viruses in current and future outbreaks. Thus, SHINEv2 can provide critical information to inform public health responses, and it fills a major gap in point-of-need diagnostics. At the population level, SHINEv2 could be used to prioritize testing and vaccine rollout in highly-affected communities or to select subsets of samples for further viral sequencing. SHINEv2 could also assist clinicians in selecting the right treatment (e.g. monoclonal antibody cocktails) for patients with severe CO...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants

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