DNA Dynamics in Drosophila: Detecting Chromosomal Translocations Using High-Throughput Genome-Wide Translocation Sequencing
Do, Vicki
2023
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Abstract: Chromosomal translocations are structural genome variants (SGVs) that arise when two non-homologous chromosomes exchange genetic material through the repair of two separate DNA double-strand breaks (DSBs). The repair of these breaks can lead to the formation of fusion genes or alterations in gene expression, which has been implicated in the development of many malignancies and diseases, ... read moreparticularly cancer. The repair process for these breaks involves either classical non-homologous end joining (c-NHEJ) or alternative end-joining (alt-EJ), with the latter being more mutagenic and utilizing microhomology (MH) sequences aligned by polymerase theta (POLQ). The literature on translocation formation has presented conflicting data regarding the underlying mechanisms and factors involved in this process. In humans, translocation formation appears to be driven by c-NHEJ whereas in mice, alt-EJ plays a more dominant role. High-throughput genome-wide translocation sequencing (HTGTS) is a technique that has been used to study translocations in mammalian cells and allows for the identification of translocation junctions between two different genomic loci termed “bait” and “prey” sites. This project attempts to adopt the HTGTS workflow for Drosophila melanogaster, utilizing Cas9 and single-guide RNAs (sgRNAs) with low specificity to generate translocations at various sites throughout the fly genome. Validation of efficient sgRNAs has been achieved through in vitro cutting assays, and these guide constructs have been successfully cloned into the pU6-BbsI- chiRNA plasmid expression vector. The next steps will be to continue transfecting these plasmids containing the sgRNAs in Cas9-expressing Drosophila S2 cells for Cas9 cleavage, and the translocation junctions will be sequenced using high-throughput amplicon sequencing and analyzed using a published HTGTS bioinformatic pipeline. This work will be done in different DNA repair backgrounds to elucidate the contributions of each in translocation formation as well as identify the role of POLQ and alt-EJ in mediating these events. The same sgRNA expression plasmids were also injected into early Drosophila embryos endogenously expressing Cas9 to investigate the potential for inducing breaks and translocations in their genomes, in parallel with the HTGTS project. In an additional but related study, a previously established plasmid-based system was adapted as a proxy to investigate the preferences for types of chromosomal rearrangements in Drosophila.
Thesis (B.S.)--Tufts University, 2023.
Submitted to the Dept. of Biology.
Advisors: Mitch McVey, Nick Woodward.read less - ID:
- 0c484036b
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