Emmeline L. Blanchard, Daryll Vanover, Swapnil Subhash Bawage, Pooja Munnilal Tiwari, Laura Rotolo, Jared Beyersdorf, Hannah E. Peck, Nicholas C. Bruno, Robert Hincapie, Frank Michel, Jackelyn Murray, Heena Sadhwani, Bob Vanderheyden, M. G. Finn, Margo A. Brinton, Eric R. Lafontaine, Robert J. Hogan, Chiara Zurla & Philip J. Santangelo

Nature Biotechnology (2021) 03 February 2021

Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.

See: https://www.nature.com/articles/s41587-021-00822-w

Fig. 1: a, Schematic representation of A/WSN/33 genome showing the localization of the crRNA guides. b, A549 cells were infected with IAV A/WSN/33 at MOI = 0.01. Twenty-four hpi, cells were transfected with Cas13a or Cas13a-NLS and 1:50 mol ratio of WSN33_PB1 or NTCR guides. Two-way ANOVA with Sidak’s multiple comparisons, where **P = 0.0001 and ****P < 0.0001. c, A549 cells were infected with A/WSN/33. Twenty-four hpi, cells were transfected with either Cas13a or Cas13a-NLS, dead Cas13a, dead Cas13a-NLS mRNA or an equal molar amount of GFP mRNA with a 1:50 mol ratio of WSN33_PB1_g2 or NTCR guides. Two-way ANOVA with Sidak’s multiple comparisons, where no significant differences were found. d, A549 cells were infected with A/WSN/33. Twenty-four hpi, cells were transfected with either Cas13a or Cas13a-NLS, deadCas13a, dead Cas13a-NLS mRNA or an equal molar amount of GFP mRNA with a 1:50 mol ratio of WSN33_PB1_m5 or NTCR guides. Two-way ANOVA with Sidak’s multiple comparisons, where ***P = 0.0006 and ****P < 0.0001. Means and standard deviations are shown in gray, with n = 6 biological replicates. WSN/33_PB1 gene copy numbers were normalized by NTCR values. NS, not significant.