Fig. 3.

Packaging and delivery of CRISPR-Cas9 via ARMMs. a Cas9 packaging strategy. Cas9 is fused to WW domains, which interacts with PPXY motifs of ARRDC1, allowing the recruitment of Cas9 into ARMMs. b Western blotting showing WW-linked Cas9 in EVs. HEK293T cells were transfected with Cas9, 2WW-Cas9, or 4WW-Cas9 together with pCDNA3.1 or ARRDC1-HA. Extracellular vesicles were pelleted by ultracentrifugation. Both cell lysates and extracellular vesicle (EV) pellets were subjected for anti-Flag, anti-HA, anti GAPDH, and anti-flotillin Western blotting. RNAs were also extracted from EV fractions for qRT-PCR quantification of guide RNA (c). d Transfer of WW-domain linked Cas9 proteins in ARMMs to recipient cells. HEK293T cells were incubated with different ARMMs overnight, washed with PBS extensively, and subjected to Western blotting. RNAs were also extracted from the recipient cells for qRT-PCR quantification of guide RNA (e). f Flow cytometry data showing the WW-domain linked Cas9 functioned in the recipient cells, which were U2OS cell with stable expression of GFP protein. g Quantifications of flow cytometry data (f) from three independent replicates. Data were presented as the mean ± SD in the bar graphs. *p < 0.05