# High resolution dissection of oncogene enhancer networks via CRISPR screening and live-cell imaging.

> **NIH NIH R01** · STANFORD UNIVERSITY · 2022 · $439,801

## Abstract

ABSTRACT
Non-coding elements comprise 98% of the human genome. The coordination of non-coding regulatory elements
in the mammalian genome plays a pivotal role in controlling gene expression. Both experimental and
computational studies reveal that pathogenic genes involved in complex diseases, including oncogenes, are
regulated by a large number of enhancers, implying the existence of a complex interdependent regulatory
network of enhancers in modulating and maintaining expression of these genes. Genome-Wide Association
Studies (GWAS) reveal that non-coding regulatory elements, including enhancers, are hotspots for the genetic
predisposition to disease. To determine causal relationships between chromatin architecture and gene
transcription, perturbation in a biological system is necessary. Recent advances in CRISPR-based genome
engineering and live cell imaging technologies have enabled new techniques for ultrahigh resolution interrogation
of the function of various genome regulatory elements and how they relate to gene expression. In preliminary
studies in our lab, we performed a targeted CRISPR interference (CRISPRi) based screen to study how the 7
MYC enhancers present in K562 cells work together to co-regulate this oncogene. We created a library with
>87,000 pairs of gRNAs targeting the MYC enhancers to understand the epistatic network of gene regulation
underlying MYC expression. We found that when a subset of enhancer pairs were targeted together, they
exhibited a more dramatic than expected reduction in growth rate. We developed a model that divides MYC
enhancers into 2 layers that work together with varying degrees of efficiency to co-regulate MYC expression in
K562 cells. Here, we seek to expand these preliminary results to examine additional oncogenes and perform
these experiments in additional cell types. In addition, we will combine perturbation of oncogene enhancers with
CRISPR-based live cell imaging (termed CRISPR LiveFISH), that allows for the dynamic imaging of multiple
genomic loci, mRNA, and protein components in living cells. In Aim 1, we will develop an ultrahigh-resolution
multiplexed CRISPRi/a tiling screens platform to dissect enhancer interactions of different oncogenes in different
cancer cell lines. We will perform multiplexed CRISPRi/CRISPRa screens to inhibit or activate pairs of enhancers
with an ultrahigh spatial resolution (~20bp) controlling four oncogenes (MYC, CCND, BCL2, PDE4DIP) in K562
and HeLa cells. In Aim 2, we will characterize the dynamic real-time interactions between transcriptional
coactivators, mediators, multiple enhancers, promoters, and RNA transcription during CRISPRi/a-mediated
perturbation. We will monitor real-time dynamics of different enhancers, promotors, RNA transcription, and the
transcriptional coactivator proteins BRD4, IRF1, and Gata4 using LiveFISH with and without enhancer
perturbation. Altogether, we seek to apply new CRISPR technologies developed in our lab to create a model of
how ...

## Key facts

- **NIH application ID:** 10522013
- **Project number:** 1R01CA266470-01A1
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Lei Stanley Qi
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $439,801
- **Award type:** 1
- **Project period:** 2022-08-01 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10522013

## Citation

> US National Institutes of Health, RePORTER application 10522013, High resolution dissection of oncogene enhancer networks via CRISPR screening and live-cell imaging. (1R01CA266470-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10522013. Licensed CC0.

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