An unbiased survey of distal element-gene regulatory interactions with direct-capture targeted Perturb-seq

A major challenge in human genetics is to identify all distal regulatory elements and determine their effects on target gene expression in a given cell type. To this end, large-scale CRISPR screens have been conducted to perturb thousands of candidate enhancers. Using these data, predictive models have been developed that aim to generalize such findings to predict which enhancers regulate which genes across the genome. However, existing CRISPR methods and large-scale datasets have limitations in power, scale, or selection bias, with the potential to skew our understanding of the properties of distal regulatory elements and confound our ability to evaluate predictive models. Here, we develop a new framework for highly powered, unbiased CRISPR screens, including an optimized experimental method (Direct-Capture Targeted Perturb-seq (DC-TAP-seq)), a random design strategy, and a comprehensive analytical pipeline that accounts for statistical power. We applied this framework to survey 1,425 randomly selected candidate regulatory elements across two human cell lines. Our results reveal fundamental properties of distal regulatory elements in the human genome. Most element-gene regulatory interactions are estimated to have small effect sizes (<10%), which previous experiments were not powered to detect. Most cis -regulatory interactions occur over short genomic distances (<100 kb). A large fraction of the discovered regulatory elements bind CTCF but do not show chromatin marks typical of classical enhancers. Housekeeping genes have similar frequencies of distal regulatory elements compared to other genes, but with 2-fold weaker effect sizes. Comparisons to the predictions of the ENCODE-rE2G model suggest that, while performance is similar across two cell types, new models will be needed to detect elements with weaker effect sizes, regulatory effects of CTCF sites, and enhancers for housekeeping genes. Overall, this study describes the first unbiased, perturbation-based survey of thousands of distal regulatory element-gene connections, and provides a framework for expanding such efforts to build more complete maps of distal regulation in the human genome.