Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

• Published in: Cell 2018-11, Vol.175 (4), p.1141-1155.e16
• Main Authors: Wroblewska, Aleksandra, Dhainaut, Maxime, Ben-Zvi, Benjamin, Rose, Samuel A., Park, Eun Sook, Amir, El-Ad David, Bektesevic, Anela, Baccarini, Alessia, Merad, Miriam, Rahman, Adeeb H., Brown, Brian D.
• Format: Article
• Language: English
• Subjects: Animals; antibodies; barcoding; cancer; CRISPR; CRISPR-Cas Systems; DNA; epitopes; Epitopes - chemistry; Epitopes - classification; Epitopes - genetics; Flow Cytometry - methods; functional genomics; gene editing; genes; Genomics - methods; HEK293 Cells; Humans; Immunophenotyping - methods; interferon gamma pathway; Jurkat Cells; mass cytometry; Mass Spectrometry - methods; Mice, Inbred BALB C; neoplasm cells; neoplasms; phenotype; pooled screen; protein barcodes; protein content; Proteome - chemistry; Proteome - classification; Proteome - genetics; screening; single cell analysis; Single-Cell Analysis - methods; T cells; THP-1 Cells; tumor immunology
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2018.09.022

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). Pro-Code-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. [Display omitted] •Generated 100s of genetic barcodes detectable as proteins (Pro-Codes)•Pro-Codes provide a single-cell resolution means for vector and cell tracking•Pro-Codes enable high-dimensional phenotyping for CRISPR screens•This approach identified roles for Rtp4, Psmb8, and Socs1 in cancer immune editing Protein-level genetic barcodes enable single-cell high-dimensional phenotyping by mass cytometry in CRISPR screens.