Modulation of DNA repair pathway after CRISPR/Cas9 mediated Double Stranded Break

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dc.contributor.authorSeo, Jooheonen
dc.contributor.committeechairLee, Kihoen
dc.contributor.committeememberRhoads, Michelleen
dc.contributor.committeememberEaly, Alan D.en
dc.contributor.departmentAnimal and Poultry Sciencesen
dc.date.accessioned2017-02-02T09:00:16Zen
dc.date.available2017-02-02T09:00:16Zen
dc.date.issued2017-02-01en
dc.description.abstractThe CRISPR/Cas9 system has become the predominant tool for genome editing. Targeted modifications can be introduced while repairing double strand breaks (DSBs), induced by the CRISPR/Cas9 system. The DSB is repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR), and the repair is commonly processed through NHEJ because it is the dominant repair pathway in most cell types. The goal of this study is to modulate DNA repair system of somatic cells to increase the frequency of homology-directed repair (HDR) through HR by chemical treatment and the frequency of NHEJ by serum starvation. CRISPR/Cas9 systems targeting RAG2 gene and donor DNA to replace endogenous RAG2 were transfected into porcine fetal fibroblast (PFF) cells and the cells were treated with various chemicals that were known to inhibit NHEJ or stimulate HR. Among the chemical treated groups, cells treated with thymidine showed an average of 5.85-fold increase in HDR compared to the control group; the difference ranged from 1.37 to 9.59. There was no positive effect on the frequency of HDR after treating transfected cells with other chemicals. Placing PFFs under low amount of serum (serum deprivation) could enrich the cells in G0/G1 phase, but there was little difference in the frequency of NHEJ. Our results indicate that modulating DNA repair pathways during CRISPR/Cas9-mediated gene targeting could change the outcome of the targeted events.en
dc.description.degreeMaster of Scienceen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:9396en
dc.identifier.urihttp://hdl.handle.net/10919/74884en
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectCRISPR-Cas9 systemen
dc.subjectgenetic engineeringen
dc.subjectmodified cell lineen
dc.subjectgene targetingen
dc.subjectrepair systemen
dc.subjectcell cycleen
dc.titleModulation of DNA repair pathway after CRISPR/Cas9 mediated Double Stranded Breaken
dc.typeThesisen
thesis.degree.disciplineAnimal and Poultry Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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