A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance

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1. Version published to 10.1089/crispr.2022.0048

   Apr 1, 2023
2. 

   ScreenIT

   Mar 21, 2022

   SciScore for 10.1101/2022.03.17.22272589: (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.

   Table 2: Resources

   Recombinant DNA
   Sentences	Resources
   Gp32 and uvsX were cloned into the pET28a backbone using Type IIS assembly (Engler et al., 2008).	pET28a suggested: RRID:Addgene_139598)
   Plasmid #163912); pET28a- uvsX-H6	pET28a- suggested: None
   (Plasmid #163913); and pET28a-MH6-uvsY (Plasmid #163914).	pET28a-MH6-uvsY suggested: None
   Expression and purification of Cas enzymes: LwaCas13a expression and purification: LwaCas13a was expressed from pC013-His6-Twinstrep-SUMO-LwaCas13a (Addgene: #90097) and purified as previously described (Patchsung et al., 2020).	pC013-His6-Twins…

   SciScore for 10.1101/2022.03.17.22272589: (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.

   Table 2: Resources

   Recombinant DNA
   Sentences	Resources
   Gp32 and uvsX were cloned into the pET28a backbone using Type IIS assembly (Engler et al., 2008).	pET28a suggested: RRID:Addgene_139598)
   Plasmid #163912); pET28a- uvsX-H6	pET28a- suggested: None
   (Plasmid #163913); and pET28a-MH6-uvsY (Plasmid #163914).	pET28a-MH6-uvsY suggested: None
   Expression and purification of Cas enzymes: LwaCas13a expression and purification: LwaCas13a was expressed from pC013-His6-Twinstrep-SUMO-LwaCas13a (Addgene: #90097) and purified as previously described (Patchsung et al., 2020).	pC013-His6-Twinstrep-SUMO-LwaCas13a suggested: None
   PsmCas13b expression and purification: Escherichia coli BL21 (DE3) transformed with pC0061-His6-Twinstrep-SUMO-PsmCas13b (Addgene: #115211) were grown in LB media containing 25 µg/mL ampicillin, and the protein expression induced by the addition of 500 µM IPTG at 16 °C for overnight.	pC0061-His6-Twinstrep-SUMO-PsmCas13b suggested: None
   Software and Algorithms
   Sentences	Resources
   Expression and purification of Cas enzymes: LwaCas13a expression and purification: LwaCas13a was expressed from pC013-His6-Twinstrep-SUMO-LwaCas13a (Addgene: #90097) and purified as previously described (Patchsung et al., 2020).	Addgene suggested: (Addgene, RRID:SCR_002037)
   Four picomoles of the purified dsDNA crRNA template were used in in vitro transcription reactions using RiboMAX Large Scale RNA Production System–T7 (ProMega) or MEGAshortscript™ T7 Transcription Kit (Invitrogen).	MEGAshortscript™ suggested: None
   Gels were imaged on ImageQuant™ LAS 4000 (GE Healthcare); quantifications of produced crRNAs were performed by measuring band densitometries using Fiji ImageJ software (Schindelin et al., 2012) and comparing to RNA standards.	Fiji ImageJ suggested: None
   Retrieved sequences were aligned using MAFFT (Katoh & Standley, 2013).	MAFFT suggested: (MAFFT, RRID:SCR_011811)
   The four consensus sequences along with the SARS-CoV- 2 isolate Wuhan-Hu-1 NCBI reference genome (Accession ID NC_045512.2) were re-aligned using MAFFT and visualized in SnapGene software (Insightful Science).	SnapGene suggested: (SnapGene, RRID:SCR_015052)
   Exclusivity evaluation of designed crRNAs: crRNA spacer sequences and their complementary sequences were searched against a BLAST database of Betacoronavirus nucleotide sequences (	BLAST suggested: (BLASTX, RRID:SCR_001653)
   Betacoronavirus BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=Betacoronavirus), with the maximal number of target sequences set to 5000.	https://blast.ncbi.nlm.nih.gov/Blast.cgi suggested: (TBLASTX, RRID:SCR_011823)
   The resulted alignments were visualized and inspected using JalView (Aksamentov et al., 2021).	JalView suggested: (Jalview, RRID:SCR_006459)

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   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).

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   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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   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.
   • Thank you for including a protocol registration statement.

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

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3. 

   Version published to 10.1101/2022.03.17.22272589 on medRxiv

   Mar 18, 2022