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

## TL;DR

The paper introduces TESLA-seq, a method combining CRISPR activation and single-cell RNA sequencing to map enhancer-gene relationships, revealing regulatory connections around the PHOX2B gene in neuroblastoma.

## Contribution

TESLA-seq enables high-sensitivity mapping of enhancer-gene interactions using CRISPRa and single-cell RNA-seq.

## Key findings

- TESLA-seq identified 60 regulators for 30 genes around the PHOX2B locus.
- Many enhancer-gene interactions were active in tissues outside the experimental system.
- CRISPRa tiling screens revealed hundreds of growth-modulating elements near PHOX2B.

## Abstract

Enhancers are known to spatiotemporally regulate gene transcription, yet the identification of enhancers and their target genes is often indirect, low resolution, and/or assumptive. To identify and functionally perturb enhancers at their endogenous sites, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma, and found many CRISPRa-responsive elements (CaREs) that alter cellular growth. To determine CaRE target genes, we developed TESLA-seq (targeted single-cell activation), which combines CRISPRa screening with targeted single-cell RNA sequencing and enables 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-related regulatory elements, we found many CaREs and target connections normally active only in other tissues. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system.

•CRISPRa tiling screen reveals hundreds of growth-modulating elements surrounding PHOX2B•TESLA-seq finds gene targets of dozens of candidate CRISPRa-responsive elements (CaREs)•Validated regulatory CaRE-gene interactions and profiled their genomic features•Integrated epigenomics data identify tissues where CaRE-gene pairs are active

CRISPRa tiling screen reveals hundreds of growth-modulating elements surrounding PHOX2B

TESLA-seq finds gene targets of dozens of candidate CRISPRa-responsive elements (CaREs)

Validated regulatory CaRE-gene interactions and profiled their genomic features

Integrated epigenomics data identify tissues where CaRE-gene pairs are active

Vučićević et al. describe TESLA-seq, which combines pooled CRISPR activation with targeted single-cell RNA-seq to map enhancer-gene connections at high sensitivity. TESLA-seq was applied across a genomic region surrounding the PHOX2B gene, a key transcription factor affecting the growth of neuroblastoma cells, to reveal 60 regulators for 30 genes.