Fig. 5: RNA delivering T4-AVVs.

a Schematic illustration of T4-AVVs carrying siRNA and mRNA on the capsid surface as complexes of Cas9-Soc. These AVVs can carry out multiple genetic operations upon delivery; gene silencing (siRNA cargo), genome editing (RNP cargo), and gene expression (DNA and mRNA cargo). b Agarose gel showing the binding of siRNA to T4(gRNA-GFP)-[Cas9-Soc] capsids at increasing ratios of siRNA molecules to Soc binding sites. The positions of the packaged gRNA-GFP plasmid and the displayed siRNA are indicated. c Silencing (knock-down) of GFP expression in 293T cells treated with T4(gRNA-GFP)-[GFPsiRNA-Cas9-Soc]-AVVs (GFPsiRNA-AVVs). The control T4(gRNA-GFP)-[NCsiRNA-Cas9-Soc]-AVVs (NCsiRNA-AVVs) carry a nonspecific siRNA. Bar = 50 μm. d Western blotting quantification using a GFP-specific mAb showing suppression of GFP protein levels by GFPsiRNA-AVVs at 48 and 72 h post-transduction (n = 3). e Simultaneous silencing of two different genes by incorporating two siRNAs into the same AVV, one targeted to GFP gene and the other to endogenous GAPDH gene (n = 3). f AAVS1 indel frequencies of cells treated with T4(U6pro-AAVS1gRNA-CMVpro-GFP)-[siRNA-Cas9-Soc]-AVVs at increasing siRNA ratios (n = 3). The inset at the top right shows AAVS1 gene disruption using T7E1 assay. g Agarose gel showing the binding of GFPmRNA to T4(mCherry)-[Cas9-Soc] capsids at increasing ratios of mRNA molecules to Soc binding sites. The positions of the packaged mCherry plasmid and the displayed GFPmRNA bands are indicated. h Dual expression of AVV-packaged mCherry plasmid DNA and AVV-displayed GFPmRNA in the same cell. Bar = 50 μm. i Genome editing at AAVS1 locus by T4(U6pro-AAVS1gRNA-CMVpro-mCherry)-[GFPmRNA-Cas9-Soc]-AVVs at increasing mRNA ratios (n = 3). Values represent mean with SD.