Fig. 2: Cas13d strongly inhibits the human coronaviruses HCoV-229E and HCoV-OC43, singly and together.

a The genome structure of 229E. Includes the locations of the 20 crRNAs targeting the RdRp gene and the 20 crRNAs targeting the N gene. b–d Inhibition of 229E by Cas13d. At 48 hpi, the virus titer in the supernatant (b) was measured by RT-qPCR. Viral RNA abundance in cell lysates was quantified using two sets of primers targeting RdRp (c) and N (d) gene, respectively; n = 3, t = 3. e, f Cell viability after 229E challenge was measured using the Incucyte live imaging system. e Incucyte images showing brightfield and fluorescence overlay for selected time points. Dead cells are in green and Cas13d+ cells are in red; n = 3. f Quantification of the signal from dead cells over time; n = 3 wells per group, 9 images per well, per time point. Data presented as means ± SEM. g The genome structure of OC43 and the locations of the 10 N gene-targeting crRNAs. h Inhibition of OC43 by Cas13d. At 48 hpi, the viral genome copies in the supernatant were measured by RT-qPCR; n = 2, t = 3. i Phylogenetic tree of representative strains of SARS-CoV-2, 229E, OC43. The outer ring shows coverage by each of the top crRNAs targeting these three coronaviruses. j–l Co-infection of 229E and OC43 in MRC-5 cells expressing different combinations of crRNAs. j Schematic depicting the strategy to protect against both 229E and OC43 simultaneously. At 48 hpi, the 229E (k) or OC43 (l) virus titer in the supernatant was measured by RT-qPCR; n = 3, t = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate. All source data in this figure are provided as a Source data file. P values for virus-targeting crRNAs (refer to Supplementary Data 3) are relative to NT. APN aminopeptidase N, SA:9-O-ac 9-O-acetyl-modified sialic acid.