Generating Stable B2M-KO AM-MSCs

Human genomic B2M sequences were analyzed and selected using the web tool Benchling (https://benchling.com/). B2M-specific CRISPR-Cpf1 expression vector was constructed by cloning the annealed oligomers (5′-agatCCGATATTCCTCAGGTACTC-3′ and 5′-aaaaGAGTACCTGAGGAATATCGG-3′) into a pY108 lentiviral vector (Addgene, plasmid #84739). Infectious lentiviral particles were produced as described previously and were precipitated using Lenti Concentrator (Origene, Rockville, Maryland, US) according to the manufacturer’s protocol [24 (link)]. To produce stable B2M-KO-AM-MSCs, resuspended lentivirus in culture media were added to AM-MSCs and were incubated for 24 h in the culture medium. The cell culture medium was replaced with a fresh medium containing 4 μg/ml puromycin and incubation continued for a further 24 h. AM-MSCs in which B2M was knocked out and that did not express MHC I was selected with a BD FACSAria™ III Cell Sorter. B2M-KO was accessed by an Indel sequencing primer (Table S1).