# High-throughput characterization of epigenetic context on DNA double strand break repair dynamics

> **NIH NIH F30** · JOHNS HOPKINS UNIVERSITY · 2021 · $51,036

## Abstract

Project Summary
The survival and fitness of complex multicellular organisms depend on the successful propagation and
maintenance of our “genetic code” in deoxyribonucleic acid (DNA). However, the genomic integrity of our cells
is under constant assault, so effective DNA repair processes are absolutely essential. There are many forms of
DNA damage, but double strand breaks (DSBs) are common and the most toxic. As a consequence, DSBs are
repaired by multiple redundant and/or competing repair pathways. After DSBs are formed, how the cell detects
the damage, recruits specific DNA repair effectors, and decides pathway choice are important questions. While
recent literature has made great strides in these directions, the spatiotemporal dynamics of repair factor
recruitment at single DSBs have not been sufficiently explored. This has been a challenging question to address
because no method has been able to generate pure, sequence specific DSBs with the temporal resolution that
matches the rapidity of the DSB damage response. We recently developed a very fast, light-inducible
CRISPR/Cas9 system that fulfills this purpose. Preliminary data have demonstrated that our system efficiently
induces DSBs within seconds, and we have used this system to investigate the dynamics of repair factor
recruitment primarily through imaging assays. However, we can only interrogate a single break site at a time due
to the technical limitations of imaging. Enabled by the technological advances of our new method, I propose to
study the dynamics of DSB response in high-throughput and on genomic coordinates. My proposed research
strategy is to 1) develop time-resolved chromatin immunoprecipitation sequencing (ChIP-seq) assays to track
the recruitment or departure of several DNA damage response (DDR) factors after synchronized DSBs at a
validated target sequence, 2) establish a platform for generating hundreds of sequence-specific DSBs, followed
by ChIP-seq to correlate the spatiotemporal dynamics of DDR factors with prior epigenetic and transcriptional
states, and 3) induce perturbations with transcription inhibitors and CRISPR-based gene activation or repression
(CRISPRa or CRISPRi, respectively) to establish cause and effect relationships between transcription and DDR
factor recruitment. Together, these studies will further elucidate how cells physiologically respond to genotoxic
insults and validate a novel platform for investigating how those responses are crippled by disease, especially
in cancer and inherited disorders of DNA repair. Furthermore, improved understanding of how cells repair DSBs
will help enhance the safety and efficacy of genome editing agents like CRISPR/Cas9.

## Key facts

- **NIH application ID:** 10223883
- **Project number:** 5F30CA254160-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Roger Zou
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $51,036
- **Award type:** 5
- **Project period:** 2020-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10223883

## Citation

> US National Institutes of Health, RePORTER application 10223883, High-throughput characterization of epigenetic context on DNA double strand break repair dynamics (5F30CA254160-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10223883. Licensed CC0.

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