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

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http://urn.fi/URN:NBN:fi:hulib-202012165272
Title: Creating CRISPR-Cas9 genome edited iPSC lines to model a patient-specific mutation in mitochondrial disease
Author: Jalkanen, Nelli
Other contributor: Helsingin yliopisto, Lääketieteellinen tiedekunta
University of Helsinki, Faculty of Medicine
Helsingfors universitet, Medicinska fakulteten
Publisher: Helsingin yliopisto
Date: 2020
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202012165272
http://hdl.handle.net/10138/323231
Thesis level: master's thesis
Degree program: Translationaalisen lääketieteen maisteriohjelma (Translational Medicine)
Master's Programme in Translational Medicine
Magisterprogrammet i translationell medicin
Specialisation: Neuroscience and psychobiology
Neuroscience and psychobiology
Neuroscience and psychobiology
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.
Subject: CRISPR-Cas9
genome editing
iPSC
disease modelling
mitochondrial aminoacyl tRNA-synthetase
seryl tRNA-synthetase
mitochondrial disease


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