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Creating CRISPR-Cas9 genome edited iPSC lines to model a patient-specific mutation in mitochondrial disease

Show simple item record 2020-12-16T10:34:41Z 2020-12-16T10:34:41Z 2020-12-16
dc.title Creating CRISPR-Cas9 genome edited iPSC lines to model a patient-specific mutation in mitochondrial disease en
ethesis.faculty Lääketieteellinen tiedekunta fi
ethesis.faculty Faculty of Medicine en
ethesis.faculty Medicinska fakulteten sv
ethesis.faculty.URI Helsingin yliopisto fi University of Helsinki en Helsingfors universitet sv
dct.creator Jalkanen, Nelli
dct.issued 2020
dct.language.ISO639-2 eng
dct.abstract Mitochondrial aminoacyl tRNA-synthetases (mt-aaRS) catalyse the charging of tRNAs with their cognate amino acids in mitochondria. Mutations in mt-aaRS cause tissue-specific mitochondrial diseases, especially affecting tissues with high energy expenditure like the nervous system, heart, and kidneys. However, disease mechanisms for the heterogeneous group of diseases have not yet been fully elucidated. Harnessing CRISPR-Cas9 genome editing in induced pluripotent stem cells (iPSC) provides an opportunity to model mt-aaRS mutations in vitro and investigate the effects of individual mutations on cellular phenotype. SARS2 encodes mitochondrial seryl tRNA-synthetase, and its c.1347 G>A mutation causes severe childhood-onset progressive spastic paresis. Here, CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology directed repair (HDR) the genome of iPSC and knock-in the patient mutation. Guide RNAs were designed and tested for efficiency before electroporation into wild type iPSC. Clonal cell lines were made by low-density seeding and manual colony picking. The expression of pluripotency markers was measured by RT-qPCR. RT-qPCR and Western blot measured SARS2 mRNA expression and protein level respectively. The success and precision of genome editing were analysed by Sanger sequencing, comparing the performance of the different guide RNAs, and screening regions of potential off-target genome editing. Two genome-edited iPSC lines with the SARS2 c.1347 G>A mutation were successfully generated to model the patient mutation. The iPSC lines expressed pluripotency markers and contained no off-target genome editing and modelled the patient’s decrease in SARS2 protein level and mRNA expression. More evidence of differentiation ability is needed before differentiation into the affected cell type (motor neurons) and further disease modelling. The efficiency of CRISPR-Cas9 for genome editing, especially harnessing HDR in iPSC, is an area of future research. en
dct.subject CRISPR-Cas9
dct.subject genome editing
dct.subject iPSC
dct.subject disease modelling
dct.subject mitochondrial aminoacyl tRNA-synthetase
dct.subject seryl tRNA-synthetase
dct.subject mitochondrial disease
dct.language en
ethesis.language englanti fi
ethesis.language English en
ethesis.language engelska sv
ethesis.supervisor Tyynismaa, Henna
ethesis.thesistype pro gradu -tutkielmat fi
ethesis.thesistype master's thesis en
ethesis.thesistype pro gradu-avhandlingar sv
dct.identifier.ethesis E-thesisID:b2d4f284-930f-401d-8eb5-e321294feb26
ethesis.degreeprogram.URI none
ethesis-internal.timestamp.reviewStep 2020-11-04 15:32:47:837
dct.identifier.urn URN:NBN:fi:hulib-202012165272
dc.type.dcmitype Text Neurogenetics und
ethesis.facultystudyline Neuroscience and psychobiology fi
ethesis.facultystudyline Neuroscience and psychobiology en
ethesis.facultystudyline Neuroscience and psychobiology sv
ethesis.mastersdegreeprogram Translationaalisen lääketieteen maisteriohjelma (Translational Medicine) fi
ethesis.mastersdegreeprogram Master's Programme in Translational Medicine en
ethesis.mastersdegreeprogram Magisterprogrammet i translationell medicin sv

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