From variants to cytokines: comprehensively characterizing how cells respond to perturbations

Abstract

Cells can be perturbed by a changing environment or a mutation in their genome. Therefore, the high-throughput single-cell dissection of both genotype-phenotype and signal-response relationships is pivotal to understanding cell function and disease. Here, we present two complementary platforms that leverage barcoding strategies to this end. The first platform, Variant In Situ Sequencing (VIS-seq), co-expresses protein-coding variants and linked circularized RNA barcodes that are easily sequenced in situ. This approach enables the simultaneous optical profiling of thousands of variants in a gene by mapping variant identity to cellular phenotypes. Applying VIS-seq to >3000 variants of LMNA and PTEN in >13 million sequenced cells from diverse cell types revealed detailed structure–function relationships, including variant-induced changes in protein localization, cell and nuclear morphology, and biochemical activity, illuminating mechanisms underlying laminopathies and PTEN-associated disorders. The second platform, CellCode, harnesses retroviral cell barcoding to uniquely tag individual clones, permitting pooled interrogation of cell-extrinsic perturbations. Integrated with single-cell transcriptomics and functional assays for cell growth and differentiation, CellCode generated an ex-vivo contextual atlas of the effects of 28 cytokines in mouse CD8+ T-cells. VIS-seq and CellCode are robust, highly scalable methodologies to interrogate the effect of genetic and environmental inputs on cellular biology.