Skip to main content

Advertisement

Fig. 3 | BMC Biotechnology

Fig. 3

From: Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies

Fig. 3

Generation and selection of HEK 293 cells expressing affinity-matured antibodies. a Overall strategy for antibody affinity maturation. HEK 293 cells expressing the initial Ab are subjected to CRISPR-X mutagenesis. Cells expressing variant antibodies of higher avidity are enriched using Stringent Tetramer-Associated Magnetic Enrichment (S-TAME) and expanded in vitro (R for “ enriched population”, subscript n for round of mutation/selection). Enriched cells are separated by FACS into tetramer positive-staining (R+) and tetramer negative-staining (R-) populations. Multiple rounds of mutation/selection can be performed successively as indicated. b Staining of A2Ab-expressing HEK 293 cells with 4A2-tetramers or 3A2/1B7 tetramers as marked after 3 successive transfections for CRISPR-X mutagenesis. Results shown are before the S-TAME step. c Staining of cells with tetramer 3A2/1B7 after S-TAME. Results are shown for cells transfected with dCas9, sgRNAs and MS2 AID*Δ (R1 cells, left panel), AID*Δ alone (middle panel) and sgRNA alone (right panel). d Cells from the R1 population staining positive with the 3A2/1B7 tetramer were isolated by FACS (R1+ cells). Staining of these cells with tetramers 3A2/1B7 (upper left panel) and 1A2/3B7 (upper right panel) is shown. R1+ cells were subjected to a second round of mutagenesis, S-TAME and FACS selection to generate R2+ cells. Staining of R2+ cells with tetramers 3A2/1B7 (lower left panel) and 1A2/3B7 lower right panel) is shown. The number of cells within marked gates is shown between brackets as a percentage of the total cells analysed

Back to article page