Summary CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). ProCode-expressing vectors were introduced into cells and analyzed by CyTOF mass-cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells, and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single cell resolution.

L. provided samples. M.M. designed and supervised the research, analyzed data, and edited the manuscript. J.D.B. analyzed data and edited the manuscript. B.D.B. designed and supervised the research, analyzed data and wrote the manuscript. COMPETING INTERESTS J.M.S. is an employee and stockholder of Qiagen Sciences. J.D.B. holds patents related to ATAC-seq and is on the scientific advisory board for Camp4, Seqwell, and Celsee. The remaining authors have no competing interests.