Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance

Figures

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  Fig. 1. Reprogrammed pspCas13b silences Spike transcripts with high efficiency and specificity. a Schematic of pspCas13b reporter assay used to track the recognition and degradation of SARS-CoV-2 Spike RNA. b Representative fluorescence microscopy images show the silencing of Spike transcripts with 4 targeting crRNAs in HEK 293 T (upper panel) and VERO cells (lower panel). NT is a non-targeting control crRNA; Images are processed for quantification using ImageJ. Scale bar = 300 μm. Similar results were obtained in 3 independent experiments. Unprocessed representative images are provided in the Source Data file. c Quantification of silencing efficiency with various crRNAs in HEK 293 T and VERO cells. Data points in the graph are normalized mean fluorescence from 4 representative fields of view per experiment imaged in N = 3. The data are represented in arbitrary units (A.U.). Errors are SD and p-values of one-way Anova test are indicated (95% confidence interval). d crRNA dose-dependent silencing of Spike transcripts with either NT (red) or spike targeting crRNA2 (black) in 96-well containing 100 µL of media; Data points in the graph are normalized mean fluorescence from 4 representative…

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  Fig. 2. pspCas13b suppresses SARS-CoV-2 nucleoprotein (NCP) transcripts with high efficiency and specificity. a Schematic of pspCas13b reporter assay to monitor NCP silencing with various pspCas13b crRNAs. b Representative fluorescence microscopy images show the silencing efficiency of the NCP transcripts with 3 targeting crRNAs in HEK 293 T. NT is a non-targeting control crRNA. Scale bar = 300 μm. Similar results were obtained in 3 and 6 independent experiments in HEK 293 T and VERO cells, respectively. Unprocessed representative images are provided in the Source Data file. c The histograms quantify the silencing efficiency of various crRNAs in HEK 293 T (N = 3) and VERO cells (N = 6). Data points are normalized mean fluorescence from 4 representative fields of view per condition imaged. The data are represented in arbitrary units (A.U.). Errors are SD and p-values of one-way Anova test are indicated (95% confidence interval). d Western blot analysis of NCP silencing efficiency in HEK 293 T obtained with various crRNAs (N = 3; uncropped blots from 3 independent biological experiments are provided in Source data). e crRNA dose-dependent silencing of the NCP transcripts with either NT…

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  Fig. 3. pspCas13b silencing tolerates single-nucleotide mismatch with SARS-CoV-2 targets. Fluorescence-based reporter assays to assess the silencing efficiency of Spike (a) or Nucleocapsid (b) transcripts with crRNAs harbouring single-nucleotide mismatch at various location of the spacer-target interface 48 h post-transfection. Nucleotides in blue highlight the mismatch position. Data points in the graphs are mean fluorescence from 4 representative fields of view per condition imaged; N = 4. The data are represented in arbitrary units (A.U.). Errors are SD and p-values of one-way Anova test are indicated (95% confidence interval). c Representative Western blot analysis to examine the expression level of Nucleocapsid protein (3xHA tagged) in HEK 293 T cells expressing NCP-1 crRNA with single-nucleotide mismatch at various positions of the spacer-target interface 24 h post-transfection; N = 3. (See uncropped blots from 3 independent biological experiments in Source file). N is the number of independent biological replicates. Source data are provided as a Source data file.

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  Fig. 4. Silencing of SARS-CoV-2 virus replication in VERO cells. a Schematic of infection assay to assess pspCas13b-mediated suppression of SARS-CoV-2 replication in VERO cells. VERO cells were transfected with liposomes containing pspCas13b-BFP and various crRNA constructs. 48-72 h post-transfection, cells were infected with SARS-CoV-2 for 1 h and the kinetics of viral replication were assessed in supernatants collected at 1, 24, and 48 h post-infection for production of viral RNA using RT-PCR and infectious virus by quantification of the TCID50 on VERO cells. b RT-PCR and c infectivity assays to evaluate the kinetic of viral replication in VERO cells expressing either NT or NCP-targeting crRNA1 48 h post-transfection of pspCas13b and crRNAs transfection, N = 2. d RT-PCR and e infectivity assays to monitor the kinetics of viral replication in VERO cells expressing either NT or various pools of crRNA targeting NCP, Spike, NSP7, and NSP8. VERO cells were infected with a replication-competent SARS-CoV-2 48 h post-transfection of pspCas13b and crRNAs; N = 2. f RT-PCR analysis of the viral load in the supernatant of VERO cells expressing either NT, NCP-1, or various pools of crRNA targeting…

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  Fig. 5. Single crRNAs targeting the D614 genomic mutation hotspot can silence D614G variant’s RNA through the single-nucleotide mismatch tolerance. a–c Fluorescence-based reporter assay to assess the silencing efficiency of the D614G Spike transcripts with 18 tiled crRNAs harbouring a G–U (a), G–G (b) single-nucleotide mismatch, or G–C full match (c) with the target at various spacer positions; Data points in the graphs are mean fluorescence from 4 representative fields of view per condition imaged; N = 3. The data are represented in arbitrary units (A.U.). Errors are SD and p-values of one-way Anova test are indicated (95% confidence interval). N is the number of independent biological experiments. Source data are provided as a Source data file.

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  Fig. 6. Single crRNAs targeting the D614 genomic mutation hotspot can silence both ancestral and D614G variants in infected cells through single-nucleotide mismatch tolerance. a Fluorescence-based reporter assay to assess the silencing efficiency of the D614G Spike transcripts with 6 tiled crRNAs harbouring a single-nucleotide mismatch with the target at various spacer positions; Data points in the graphs are mean fluorescence from 4 representative fields of view per condition imaged; N = 3. The data are represented in arbitrary units (A.U.). Errors are SD and p-values of one-way Anova test are indicated (95% confidence interval). b Schematic of infection assays to assess pspCas13b-mediated suppression of both ancestral and D614G replication-competent SARS-CoV-2 in infected VERO cells. VERO cells were transfected with pspCas13b-BFP and various crRNA constructs. 48 h post-transfection, cells were infected with either ancestral or D614G SARS-CoV-2 for 1 h, and the kinetics of viral replication was assessed in supernatant collected at 1 h (initial viral load), 24 h, and 48 h post-infection via quantification of viral RNA in the supernatant using RT-PCR. c, e, g RT-PCR assays to evaluate…

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  Fig. 7. Schematic depiction of pspCas13b-mediated suppression of SARS-CoV-2 variants and their evolution through crRNA multiplexing and single-nucleotide mismatch tolerance. a crRNA multiplexing enables simultaneous targeting of several genomic and sub-genomic RNA locations, which limits the probability of poor silencing due to target inaccessibility (e.g. RNA folding, interaction with RNA-binding proteins in vivo). The spacer of pspCas13b mediates sequence-specific target recognition through complete base-pairing with the targeted sequence, followed by nuclease activation and target degradation. The silencing of viral RNAs alters the integrity of the viral genome, suppresses sub-genomic RNA-dependent viral protein translation, and the assembly of replication-competent viral particles within infected mammalian cells. This viral RNA targeting approach aborts viral replication cycles and inhibits the infectivity of SARS-CoV-2 virus. b The error-prone RdRP creates viral polymorphisms (or variants) through the incorporation of single-nucleotide indels, and together with host (immune system) and environmental (antiviral therapies) positive pressures, result in the emergence of new strains…