Creating CRISPR-Cas9 genome edited iPSC lines to model a patient-specific mutation in mitochondrial disease Nelli Jalkanen Master’s Thesis Master’s Degree Programme in Translational Medicine Faculty of Medicine University of Helsinki 2020 Abstract Tiedekunta – Fakultet – Faculty Koulutusohjelma – Utbildingsprogram – Degree Programme Faculty of Medicine Translational Medicine MSc. Tekijä – Författare – Author Nelli Jalkanen Työn nimi – Arbetets titel – Title Creating CRISPR-Cas9 genome edited iPSC lines to model a patient-specific mutation in mitochondrial disease Oppiaine/Opintosuunta – Läroämne/Studieinriktning – Subject/Study track Translational Medicine, specialisation in Neuroscience and Psychobiology Työn laji – Arbetets art – Level Aika – Datum – Month and year Sivumäärä – Sidoantal – Number of pages Master’s thesis October 2020 74 Tiivistelmä – Referat – 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. Avainsanat – Nyckelord – Keywords CRISPR-Cas9, genome editing, iPSC, disease modelling, mitochondrial aminoacyl tRNA-synthetase, seryl tRNA-synthetase, mitochondrial disease Ohjaaja tai ohjaajat – Handledare – Supervisor or supervisors Henna Tyynismaa PhD, Associate Professor Tiina Rasila PhD Säilytyspaikka – Förvaringställe – Where deposited Helsingin yliopiston kirjasto, Helsingfors universitets bibliotek, Helsinki University Library Muita tietoja – Övriga uppgifter – Additional information 1 Table of Contents Abstract ........................................................................................................................................... 1 Abbreviations .................................................................................................................................. 4 1. Introduction ................................................................................................................................. 7 1.1 Mitochondria ............................................................................................................................. 7 1.2 Mitochondrial tRNA-synthetases (mt-aaRS) in health and mitochondrial disease ................... 8 1.3 Mitochondrial seryl-tRNA synthetase..................................................................................... 12 1.4 SARS2 patient genetics and clinical phenotype ....................................................................... 13 1.5 Induced pluripotent stem cells in disease modelling ............................................................... 14 1.6 The creation and significance of isogenic control clonal cell lines ......................................... 16 1.7 CRISPR-Cas ........................................................................................................................... 16 1.7.1 Origins of CRISPR-Cas and developments in research ....................................................... 16 1.7.2 The CRISPR-Cas immunity mechanism and types of CRISPR-Cas ................................... 17 1.7.3 CRISPR-Cas9 in genome editing and disease modelling .................................................... 20 1.8 Aims ........................................................................................................................................ 24 2. Materials & Methods ................................................................................................................ 25 2.1 Cell culture ........................................................................................................................ 25 2.1.1 HEK293 cells ................................................................................................................ 25 2.1.2 iPSC .............................................................................................................................. 25 2.1.3 Fibroblasts ......................................................................................................................... 25 2.2 crRNA design .......................................................................................................................... 26 2.3 gRNA production .................................................................................................................... 28 2.4 gRNA testing in HEK293 cells ............................................................................................... 30 2.5 T7 endonuclease assay ............................................................................................................ 31 2.6 ssODN repair template design ................................................................................................ 32 2.7 Creating the genome edited iPSC lines ................................................................................... 33 2.7.1 Electroporation .............................................................................................................. 33 2.7.2 Subcloning .................................................................................................................... 34 2.7.3 Colony picking .............................................................................................................. 35 2.7.4 Cryopreservation of clonal cell lines............................................................................. 35 2.7.5 Thawing ........................................................................................................................ 35 2.8 Sanger sequencing............................................................................................................. 36 2.9 TOPO-Cloning .................................................................................................................. 37 2.10 Analysis and sequencing of off-target genome editing ..................................................... 38 2.11 Analyses of HDR efficiency ................................................................................................. 38 2.12 Cell collection for RT-qPCR and Western blotting .......................................................... 39 2 2.13 RT-qPCR ........................................................................................................................... 39 2.14 Western blot ...................................................................................................................... 40 2.14.1 Protein extraction .......................................................................................................... 40 2.14.2 SDS-PAGE electrophoresis and blotting ...................................................................... 40 3. Results ................................................................................................................................... 42 3.1 Two out of four crRNA were chosen for iPSC electroporation based on gene modification efficiency in HEK293 cells ........................................................................................................... 43 3.2 iPSC electroporation ............................................................................................................... 45 3.2.1 Gene modification efficiencies in the electroporated cell populations ................................ 45 3.2.2 ssODN integration and HDR efficiency in the electroporated pools ................................... 46 3.3 Clonal cell line creation .......................................................................................................... 48 3.3.1 Rates of NHEJ and HDR in the clonal cell lines ................................................................. 48 3.3.2 Two SARS2