Abstract
CRISPR screening allow us to interrogate the functions of many genes simultaneously, but comes with limitations of either being restricted to frequency analysis of bulk populations or being expensive and low throughput when used with scRNA-Seq. With FlowCodes, we adapt protein epitope barcoding for use in spectral flow cytometry, permitting tracking of genetic modifications at the single cell level both in vitro and in vivo. With spectral flow cytometry, extensive phenotypic profiling can be performed in tandem with cell tracking, allowing us to assess links between genetic manipulation and cell function. We demonstrate the unique ability of this technology to investigate rare cell populations through studies on tissue regulatory T cells (Tregs). We identify key homing molecules required for Treg trafficking through the body, critical transcriptional pathways leading to adaptation to tissue residency, and—using retrogenic T cell receptor delivery—the impact of clonality on cell fate and distribution.