Fig. 1: Antibody target deconvolution using CRISPR/Cas9 screening.

a Schematic outline of the target deconvolution process. Cells staining positive with the antibody of interest are transduced with a lentiviral sgRNA/Cas9 knockout library resulting in a heterogenous cell pool with a small population of antigen-negative cells. These cells with gene knockouts leading to lost or diminished antibody binding are isolated by FACS, the genomic DNA is extracted, and the sgRNA-encoding DNA is sequenced on the Illumina NextSeq 500 platform. Genes with sgRNAs enriched in the antigen-negative cells are identified, resulting in a proposed antibody target. b–d Representative example of flow cytometry data for mAb binding to transduced cells detected with anti-human IgG-APC. b Input cells before sort. c Pre-enriched cells after one sort with a distinct fraction of antigen-negative cells. d Sequenced cell pool after two sorts with a highly enriched fraction of antigen-negative cells. e Representative example of MAGeCK gene enrichment scores for two sort replicates, showing that the same genes are identified for both replicates. Sorts were performed in 3 to 5 replicates. Source data are provided as a Source Data file.