Fig. 7.

PTTG1IP-EV-mediated delivery of Cas9 protein.

(A) Schematic of the Cas9 stoplight reporter construct. mCherry is constitutively expressed under a CMV promoter, followed by an F2A self-cleaving peptide sequence, a Cas9-binding linker region and a stop codon. Two GFP open reading frames that are frameshifted by one or two nucleotides follow after the stop codon, separated by a F2A sequence. Upon Cas9 cleavage and indel formation, the translation reading frame will be shifted and GFP will be expressed together with mCherry (in 2/3 of the editing events). (B) Timeline of EV-transfer experiment. 24 h after donor cell transfection with Cas9 constructs and VSVG, media was replaced with fresh Opti-MEM and EVs are isolated 48 h later by SEC. Isolated EVs were then transferred to recipient cells (that were transfected with sgRNA 24 h before) in 96-well plates (3 × 10⁹ EV/ml). 24 h later media was replaced with DMEM 10% FBS and after 24 h cells are analysed for GFP expression by flow cytometry. (C) GFP positive cells quantified by flow cytometry 48 h after EV-transfer to sgRNA-transfected reporter cells. Statistical significance was tested by one-way ANOVA and Bonferroni post hoc test. Data are presented as mean +SEM, n = 3 replicates, ∗∗P < 0.01.