Generation of a CRISPR-Cas9 mediated knock-in reporter for the GRIA3 candidate gene for schizophrenia

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dc.availability.bitstreamopenaccess
dc.contributor.advisorMcCarthy, Tommie V.
dc.contributor.authorBreen, Lisa
dc.date.accessioned2023-05-17T13:53:26Z
dc.date.available2023-05-17T13:53:26Z
dc.date.issued2022-09-27
dc.date.submitted2022-09-27
dc.description.abstractGlutamatergic neurotransmission impairment is considered a major feature of the neurobiology of Schizophrenia (SZ) and implicates genes in this pathway as potential candidates for the condition. A study on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor genes found strong evidence of association between the Glutamate Ionotropic Receptor AMPA Type Subunit 3 (GRIA3) gene and SZ. Similarly, a recent report has identified a number of genes, including GRIA3, with ultra-rare disabling variants that promote SZ. The association of a rare disabling GRIA3 variant with SZ indicates that reduced expression of the gene predisposes people to SZ and suggests that increasing the expression GRIA3 could be a potential therapeutic avenue for treatment of the condition. The aim of this thesis was to establish a cell model enabling rapid analysis of GRIA3 expression. Such a model would be of high value and in addition to facilitating expression studies on GRIA3, would enable screening for new drugs that increase GRIA3 expression which could have therapeutic potential. This project aimed to modify the cell line using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR associated protein 9 (Cas9) homology directed repair (HDR) approach so that a donor reporter vector containing the Gaussia secreted luciferase gene and linked green fluorescent protein (GFP) or neomycin resistance gene (Neo) gene would be integrated directly under the control of the endogenous promoter of the GRIA3 gene, in a manner that retains intact expression of the GRIA3 protein. This donor reporter vector was successfully constructed and has significant general use as it facilitates cloning of any pair of homology arms and the insertion of a reporter cassette into any target gene via CRISPR-Cas9 HDR. Flanking GRIA3 homology arms were inserted 5’ and 3’ of the reporter cassette for CRISPR-Cas9 HDR mediated insertion into the GRIA3 locus in human U87 glioblastoma cells. Luciferase activity was monitored post-transfection and was present at low levels suggesting successful HDR events. However, the presence of the donor cassette could not be demonstrated at the GRIA3 locus. It was not possible to distinguish if the luciferase activity resulted from read through of the donor plasmid or if a low number of targeted integration events had occurred. Further work involving isolation of individual clones of the targeted U87 cells and checking for the presence of the donor at the GRIA3 locus will be necessary to resolve this question. Overall, this reporter system should be of high value for targeting other loci and can be improved further by modifications to ensure luciferase is only active when inserted into the targeted locus.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationBreen, L. 2022. Generation of a CRISPR-Cas9 mediated knock-in reporter for the GRIA3 candidate gene for schizophrenia. MRes Thesis, University College Cork.en
dc.identifier.endpage100en
dc.identifier.urihttps://hdl.handle.net/10468/14475
dc.language.isoenen
dc.publisherUniversity College Corken
dc.rights© 2022, Lisa Breen.en
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/en
dc.subjectCRISPR-Cas9en
dc.subjectGRIA3en
dc.subjectSchizophreniaen
dc.subjectKnock-in reporteren
dc.subjectHomology directed repairen
dc.titleGeneration of a CRISPR-Cas9 mediated knock-in reporter for the GRIA3 candidate gene for schizophreniaen
dc.typeMasters thesis (Research)en
dc.type.qualificationlevelMastersen
dc.type.qualificationnameMRes - Master of Researchen
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