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.

Public Health Relevance

The survival of organisms depends on the faithful maintenance of ?genetic code? in DNA despite frequent damage, especially double strand breaks (DSBs), so effective DNA repair processes are essential to mitigate harm and preserve genomic integrity. This study investigates the recruitment dynamics of essential proteins in DSB repair and explores their relationships with preexisting epigenetic and transcriptional states. This work will further elucidate how our cells physiologically respond to genotoxic insults, validate a novel platform to investigate how those responses are crippled in cancer, aging, and inherited diseases of DNA repair, and how to improve the safety and efficacy of CRISPR/Cas9 genome engineering.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1)
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Damico, Mark W
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Johns Hopkins University
Internal Medicine/Medicine
Schools of Medicine
United States
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