Team:TP-CC San Diego/Description
Project Description
Cancer is a type of genetic disease which results in an uncontrollable growth of cells; while some cancers are benign, a large number of them are malignant and can lead to death. A large proportion of human cancer is caused by the acquisition of somatic mutations across an individual's lifetime, while germline mutations inherited from parental germ cells contribute to another a small, but significant part. Though contemporary treatment methods, including radiation therapy and cytotoxic chemotherapy, have achieved substantially, they often cause severe side effects. These include fatigue, infection, numbness, nausea, and pain, due to their lack of specificity.
In 1965, extrachromosomal DNA (ecDNA) was discovered; DNA free from its traditional homes in the nucleus was documented. One study taking a look at ecDNA by means of fluorescence in situ hybridization proposed the ecDNA’s unusual number of oncogenes, but it didn’t catch enough attention because it was considered to be a rare event. Not until recently has the importance of ecDNA been revisited. The most recent study revealed that nearly 40% of oncogenes reside on ecDNA rather than the widely accepted notion that all DNA resided only on chromosomes.
Similar to chromosomal DNA, ecDNA is composed by double strands of nucleic acid but form a circular structure. More importantly, ecDNA does not have a centromere for spindle fiber binding during mitosis. This unique feature allows rapid DNA multiplication and random segregation to create high heterogeneity in daughter cells during cell proliferation, implying a possible correlative relationship between the development of tumors and a faster resistance to existing treatments.
Clustered regularly interspaced short palindromic repeats (CRISPR) technology is an effective and convenient approach for accurate gene editing mediated by Cas9 DNA nuclease and small guide RNA (gRNA). In this project, we aim to study whether specific removal of ecDNA will induce growth inhibition or death in cancer cells. To test this hypothesis, CRISPR will be used to selectively introduce DNA double strand breaks on pieces of ecDNA. First, we will identify a cancer cell line that carries ecDNA and quantify the oncogene copy number. Then, we will design and clone several gRNAs to target specific sites of the ecDNA. These gRNAs with Cas9 construct will be transfected into cancer cell line and test the cell viability. Eventually we will answer the questions posed.
