Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation

Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes remains challenging. To identify and functionally perturb enhancers at their endogenous sites, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a regulator of neuronal differentiation and neuroblastoma, revealing many CRISPRa-responsive-elements (CaREs) that alter cellular growth. To determine CaRE target genes, we developed and applied TESLA-seq (TargEted-SingLe-cell-Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing and enabled the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system.