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

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

PROVIDER: PRJNA1298184 | ENA |

REPOSITORIES: ENA

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

Project description:We developed a new framework for highly powered, unbiased CRISPR screens of distal regulatory elements, 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. Using pilot datasets from K562 and WTC11 cells, we benchmarked DC-TAP-seq against conventional scRNA-seq and TAP-seq for capture efficiency, guide detection, and knockdown assessment. Applying this optimized framework, we systematically perturbed ~1,400 randomly selected candidate regulatory elements across the two cell types and revealed key features of the human regulatory landscape. We found that most regulatory interactions have small effect sizes (<10%), occur over short genomic distances (<100 kb), and often involve CTCF-bound elements lacking classical enhancer marks. Housekeeping genes were regulated by distal elements with ~2-fold weaker effect sizes compared to other genes. Our results highlight limitations in existing CRISPR datasets and predictive models, and demonstrate the utility of DC-TAP-seq for building more comprehensive maps of distal regulation in the human genome.

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Project description:The cellular architecture of a tumor has a major impact on cancer outcome, and thus there is interest in identifying genes controlling the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a fibro-mucinous state and T-cells excluded, concomitant with upregulated TGFb and TGFb-mediated stroma activation, suggesting Tgfbr2 loss on lung cancer cells increased TGFb bioavailability and enhanced its suppressive effects on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

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