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dc.contributor.advisorKaji, Keisuke
dc.contributor.advisorTomlinson, Simon
dc.contributor.authorKaemena, Daniel Fraser
dc.date.accessioned2018-06-18T09:38:51Z
dc.date.available2018-06-18T09:38:51Z
dc.date.issued2018-06-30
dc.identifier.urihttp://hdl.handle.net/1842/31184
dc.description.abstractIn 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to ‘reprogram’ differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.subjectCRISPR/Cas9en
dc.subjectPluripotent Stem Cell reprogrammingen
dc.subjectiPSCen
dc.titleCRISPR/Cas9 genome-wide loss of function screening identifies novel regulators of reprogramming to pluripotencyen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen
dc.rights.embargodate2100-12-31en
dcterms.accessRightsRestricted Accessen


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