Regulatory elements in the genome, such as enhancers and promoters, encode sequences that orchestrate when, in which cell types, and to what extent all human genes are expressed. The activities of these sequences are fine-tuned by their endogenous chromatin context, which is crucial for quantitative, spatiotemporal regulation of gene expression. However, mapping and decoding the sequences in regulatory elements responsible for cell-type-specific gene expression remains challenging, as we lack high-throughput tools to precisely measuring the quantitative effects of sequence edits directly on gene expression in an endogenous context.

Variant-EFFECTS is broadly applicable across genes and cell types, as it is designed for use with any fluorescent readout of gene expression. It assays thousands of sorted cells for each variant and accounts for variables overlooked by existing technologies, such as editing efficiency at the endogenous locus and the presence of heterozygous cells. As a result, Variant-EFFECTS achieves >80% power to detect changes as small as 5% on gene expression and generates accurate measurements even for variants present at very low frequencies in the cell population (≥0.01% of sequencing reads). This technology unlocks our ability to explore the subtle, cell-type-specific effects of regulatory sequences on gene expression in their endogenous context.