Enrichment of DKOs with FAME strategy in HEK293T cells co-targeted for B2M and PTEN.

(A) HEK293T cells were transfected with expression plasmids for spCas9, and sgRNAs for B2M and PTEN. 5 days later, transfected cells were stained with FITC-conjugated antibody against human HLA-A,B&C. Single isolated cells with positive and negative surface MHC-I were sorted into 96-well plates. B-F. After expansion of the isolated clones, the region targeted by sgRNA was PCR amplified from genomic DNA, and submitted for Sanger sequencing. Representative chromograms were shown in the context of the target site, including PAM (GGG), for the picked clones. B, a typical wild type colony from MHC-I positive population identified with PCR sequencing; C, a typical DKO colony from MHC-I negative population with homozygous insertions identified with PCR sequencing; D, a colony (#4) from MHC-I negative population with illegible chromatogram with mixed signals; E and F, Sanger sequencing of plasmids with cloned PCR products from the same MHC-I negative colony as in D (#4) identified the exact mutations in each allele.