In vitro effect of Fas knock out on in vitro germinal center B cells

To exclude an intrinsic effect of the gene editing on cell growth,we co-cultured FasKO B cells in vitro with 40LB cells to induce a GClikephenotype in the B cells. FasWT B cells were co-cultured as acontrol with the 40LB cells. We performed next-generationamplicon sequencing on the extracted DNA from the iGC B cellson days 3 (Wednesday), 4 (Thursday), and 5(Friday) to check for mutations in the Fas gene locus thatthe crRNA had targeted. The frequency of mutations was thencompared to the flow cytometry knock-out efficiency.