Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
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Abstract
Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.
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SciScore for 10.1101/2020.11.18.389312: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Cells were routinely tested and were mycoplasma negative. Table 2: Resources
Antibodies Sentences Resources Membranes were incubated in blocking buffer (5% (w/v) skin milk powder in PBST with 0.1% Tween 20) for 1 hour at RT and probed overnight with primary antibody (anti-HA, mAb #2367, Cell Signaling Technology) at 4 °C. anti-HAsuggested: NoneBlots were washed three times in PBST with 0.1% Tween20, followed by incubation with HRP-conjugated secondary antibody (Rabbit Anti-Mouse Immunoglobulins/HRP … SciScore for 10.1101/2020.11.18.389312: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: Cells were routinely tested and were mycoplasma negative. Table 2: Resources
Antibodies Sentences Resources Membranes were incubated in blocking buffer (5% (w/v) skin milk powder in PBST with 0.1% Tween 20) for 1 hour at RT and probed overnight with primary antibody (anti-HA, mAb #2367, Cell Signaling Technology) at 4 °C. anti-HAsuggested: NoneBlots were washed three times in PBST with 0.1% Tween20, followed by incubation with HRP-conjugated secondary antibody (Rabbit Anti-Mouse Immunoglobulins/HRP #p0260, Dako) for 1 hour at RT. Anti-Mousesuggested: (Agilent Cat# P0260, RRID:AB_2636929)Experimental Models: Cell Lines Sentences Resources RNA silencing assays by transient transfection: All transfection experiments were performed in HEK293 FT and cell lines using an optimized Lipofectamine 3000 transfection protocol (Life Technologies, L3000015). HEK293suggested: NoneThe tables below summarize the transfection protocol used in 96, 24, and 6-well plates for both 293 HEK FT and VERO cells. HEKsuggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Software and Algorithms Sentences Resources Pictures were taken 48 hours (293 HEK) and 72 hours (VERO) post-transfection, and the fluorescence intensity of each image was quantified using a lab-written macro in ImageJ software. ImageJsuggested: (ImageJ, RRID:SCR_003070)All flow cytometry profiles were analyzed using FlowJo V10 software (Tree Star Inc). FlowJosuggested: (FlowJo, RRID:SCR_008520)SARS-CoV-2 RNA was converted to cDNA using the SensiFAST cDNA kit (#BIO-65053, BioLine) with 10 μL of RNA extract per reaction following the manufacturer’s instructions. BioLinesuggested: NoneData analysis: Data analyses and visualization (graphs) were performed in GraphPad Prism software version 7. GraphPad Prismsuggested: (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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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.
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