Fig. 8.

PTTG1IP-EV-mediated delivery of Cas9-sgRNA complexes.

(A) Timeline of EV-transfer experiment to reporter cells. 24 h after donor cell transfection with Cas9 constructs, sgRNA, and VSVG plasmids, media was replaced with fresh Opti-MEM and EVs isolated 48 h later by SEC. Isolated EVs were then trasferred to recipient cells in 96-well plates (3 × 10⁹ EV/ml). 24 h later media was replaced with DMEM 10% FBS and after 24 h cells analysed for GFP expression by flow cytometry. (B) GFP positive cells quantified by flow cytometry 48 h after EV-transfer to reporter cells. (C) HEK293T cells were treated with PTTG1IP-Intein-Cas9 EVs (donor cells were transfected with an RNF2-targeting sgRNA plasmid and VSVG plasmid) and gene editing assessed 48 h later by Sanger sequencing and TIDE analysis. EVs were administered at doses of (1 × 10⁸, 1 × 10⁹, and 1 × 10¹⁰ EV/ml). (D) Representative Sanger sequencing trace showing sgRNA target position and indel formation (for the 1 × 10¹⁰ EV/ml dose). The protospacer adjacent motif (PAM) site location is indicated. Statistical significance was tested by one-way ANOVA and Bonferroni post hoc test. Data are presented as mean +SEM, n = 3 replicates, ∗∗∗P < 0.001.