# High-throughput systematic characterization of regulatory element function

> **NIH NIH UM1** · STANFORD UNIVERSITY · 2020 · $1,032,594

## Abstract

Project Summary
The ENCODE project has produced high-resolution, high-quality maps of components of the `regulome' in a
set of tissues and cell lines, identifying a collection of putative regulatory elements. Our proposal aims to test
the functional relevance of these putative elements with high-throughput, pooled CRISPR screens. This
powerful platform will allow us to up-regulate, down-regulate, or mutate specific regulatory elements, and then
probe the effects of these perturbations on cell survival under normal growth conditions, and a variety of stress
conditions (oxidative stress, ricin toxicity, and nutrient deprivation) that produce differential sensitivities to gene
expression. To establish our targets, we will use ENCODE data in concert with other consortia-generated data
using an integrative analysis pipeline that leverages both correlation between element activity and gene
expression, and higher order chromatin interactions to link functional elements with potential target genes. For
~3000 genes for which we have observed that perturbation affects proliferation, we will generate multiple
libraries of ~100,000 guide RNAs for redundantly perturbing 20,000 enhancers linked to these genes through
our analysis. After identifying “hits” in this screen by sequencing the guide RNA libraries before and after
proliferation under our test conditions to observe a reduction in specific guides, we will also test combinations
of enhancer elements that may act in a cooperative or redundant fashion, exploring the functional linkages with
a specific focus on superenhancers and their sub-elements. With these phenotypic validation data in hand, we
will carry out molecular mechanistic validation by choosing 100 individual elements, and 50 combinations of
elements to generate stable cell lines with engineered genetic ablation of elements, and carry out genome-
wide molecular characterization of accessible chromatin (ATAC-seq), chromatin looping (HiChIA, a novel, high-
efficiency chromatin looping assay), and gene expression (RNA-seq). We will also generate and assess 50
lines ablating entire superenhancers and individual superenhancer elements – both individually in in
combination. For a subset of these lines, we will carry out single cell ATAC-seq to unravel effects on variations
of open chromatin within the population of cells. We will also compare results from pooled CRISPR expression
reporter assays to our method of generating edits in the native genomic context. Analyzing these data using
powerful, integrative analysis methods, scaffolded from ENCODE data, will generate global maps of the
molecular consequences of deletions at the level of chromatin and gene expression changes. These data will
be rapidly released to the community, and all techniques and cell lines will be made available to the ENCODE
consortium and to the genomics community at large. This project will deliver an immense corpus of functional
data linking regulatory elements to genes, as...

## Key facts

- **NIH application ID:** 9855027
- **Project number:** 5UM1HG009436-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** MICHAEL C BASSIK
- **Activity code:** UM1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $1,032,594
- **Award type:** 5
- **Project period:** 2017-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9855027

## Citation

> US National Institutes of Health, RePORTER application 9855027, High-throughput systematic characterization of regulatory element function (5UM1HG009436-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9855027. Licensed CC0.

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