0.45 μm pes filter

The U937 cell line was grown in RPMI 1640 media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/ml penicillin-streptomycin. The MCF7 cells were grown in DMEM media (Sigma-Aldrich) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin. The cell lines are commercially available from ATCC (U937 ATCC# CRL-1593.2 & MCF7 ATCC# HBT-22). MCF7 and U937 cells were grown to 75% confluence without FBS for 48 h prior to EV isolation. U937 cells were centrifuged 3 min at 1,200 × g to recover a cell free supernatant. Supernatants from cultures of both cell lines were used for isolation of EVs, and protease inhibitors were added (Complete Mini, Roche). The cell free supernatants were then sequentially centrifuged at 3,000 × g for 10 min and at 10,000 × g for 10 min, filtered through a 0.45 μm PES filter (VWR) and then finally centrifuged at 100,000 × g for 2 h, followed by one washing step and a second centrifugation at 100,000 × g for 2 h. Pelleted EVs were resuspended in a small amount of sterile filtered PBS supplemented with protease inhibitors (Complete Mini, Roche). Samples were stored at −80 °C and later prepared for further analysis.

Individual sgRNAs (Supplementary Table 1) were cloned into the lentiviral plasmid containing spCas9-P2A-Blast using the single guide RNA Gecko cloning protocol (https://www.addgene.org/crispr/zhang). Lentiviral vectors were transfected using Polyethylenimine (PEI) into Lenti-X (Clontech, 632180) for virus production according to the supplier’s recommendations. Virus containing supernatant was filtered through a 0.45 μm PES filter (VWR). For infection, 10⁸ mESC were plated per gelatin-coated well in a 24-well plate. After recovery for at least 4 h, cells were infected with virus diluted 2x in fresh ESCM. The next day medium was exchanged and irradiated DR4 MEF feeders were plated on top of ESC. Successful integrations were selected with 10 μg/ml blasticidin for 4 days.