Fig. 3

Characterization of LwaCas13a, PspCas13b, and CasRx activity against essential and conserved genomic region of RNA viruses. a GFP monitoring to assess the Cas13-mediated virus interference activities in Agro-infiltrated wild type N. benthamiana leaves in transient assays. Images were taken 3 days post-infiltration. NS, non-specific crRNA; Rep, replicase. b Relative fluorescence intensity quantification of leaf images. For each Cas13 variant, GFP signal intensity of each targeting crRNA (Rep-T1, Rep-T2, and Rep-T3) is shown relative to the non-targeting (NS) crRNA. Values shown as mean ± SEM (n = 3). c Western blot analysis of the abundance of the virus expressed GFP protein to confirm the Cas13-mediated TRBO-GFP virus interference. Protein blots were developed with anti-GFP and anti-HA antibodies. α-GFP, anti-GFP antibody, α-HA, anti-HA antibody. Ponceau staining served as loading control. d Quantification of the western blot data. For each Cas13 variant, the abundance of the GFP protein with the targeting crRNAs (Rep-T1, Rep-T2, and Rep-T3) is shown relative to the non-targeting (NS) crRNA. Error bars indicate SEM (n = 3). e RT-qPCR analysis of TRBO-GFP knockdown with different Cas13 variants using the three position-matched crRNAs. Transcript levels are shown relative to leaves inoculated with only TRBO-GFP vector. Values shown as mean ± SEM (n = 3)