Targeted Single-Stranded and Double-Stranded DNA Breaks using CRISPR systems in Mammalian Cells (2023)

Undergraduate: Kirti Nimmala

Faculty Advisor: Brian Diekman
Department: Joint Department of Biomedical Engineering UNC-NCSU

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Aging is an inevitable phenomenon and is a risk factor for various diseases such as cardiovascular disease, osteoarthritis, dementia, and even cancer. While it is multi-factorial, it has a common hallmark: DNA damage. DNA damage occurs from various factors such as UV radiation, carcinogens, and normal metabolism. The scale of damage is enormous with 10,000 to 100,000 DNA-damaging events occurring each day. To study DNA damage we must first cause it in cells and it is hard to determine the number of DNA breaks or their sites with traditional methods of causing damage using chemicals (like methyl methane sulfonate, etoposide) or irradiation. This study utilized CRISPR, a gene-editing tool, to generate targeted single-stranded (SSB) and double-stranded breaks (DSB) in the DNA. While previous work has utilized CRISPR to generate DSB, this is the first study, to the best of our knowledge, that used CRISPR to generate SSB. Results show that CRISPR-induced damage is detectable in the nuclear DNA using the Alkaline Comet Assay and is sensitive to the type of damage (SSB vs DSB). This study provides further validation that CRISPR systems are valuable tools to create targeted DNA breaks and have potential clinical applications in the field of cancer where cells with mutations that are associated with cancer can be selectively damaged and destroyed.

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