EV isolation from cell culture medium was performed as previously described29 (link). Briefly, ten 70–80% confluent 15 cm culture dishes of 293F cells (Invitrogen) were washed three times with PBS and then cultured in DMEM media supplemented with 10% EV-depleted FBS for 48 hours, after which conditioned media was collected and centrifuged at 2000g for 30 minutes to remove cell debris and passed through a 0.45 μm filter (LG-FPE404150S, LifeGene). EVs in this clarified supernatant were concentrated passing this material over a 100KDa centrifugal filter unit (UFC901008, Thermo Fisher Scientific) at 3000g for 20–30 minutes for three times. Retained sample was collected by washing the membrane 3× with 500 μL PBS, centrifuged twice at 4 °C, 12,000g for 30 minutes to precipitate residual debris. This supernatant was then centrifuged twice at 100,000g and 4°C for 3 hours, discarding the supernatant and resuspending the pellet in PBS after each centrifugation step. This EV fraction was then analyzed by NanoSight to determine EV size distribution and diluted and vortexed in 5 mL PBS to generate a concentrated EV solution (8.75×109 EVs/mL), which was aliquoted and stored at −80°C until aliquots were diluted for use in CRISPR-FDS liposome assays.