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Generation of Induced Pluripotent Stem Cells (Ipscs) Lines Deficient for Genes Associated with Neurodevelopmental Diseases Using CRISPR/Cas9 Technology

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Generation of Induced Pluripotent Stem Cells (Ipscs) Lines Deficient for Genes Associated with Neurodevelopmental Diseases Using CRISPR/Cas9 Technology Generation of induced pluripotent stem cells (iPSCs) lines deficient for genes associated with neurodevelopmental diseases using CRISPR/Cas9 technology Claudia De Guidi Degree project in biology, Master of science (2 years), 2021 Examensarbete i biologi 45 hp till masterexamen, 2021 Biology Education Centre and Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University Supervisor: Jens Schuster External opponent: Josefin Johansson Table of Contents Abstract ............................................................................................................................................ 3 List of abbreviations ..................................................................................................................... 4 1 Introduction ........................................................................................................................ 5 1.1 Characteristics and applications of Induced pluripotent stem cells (iPSCs). ........... 5 1.2 Neurodevelopmental disorders ............................................................................................ 6 1.3 Aim of the study ....................................................................................................................... 7 2 Materials and methods ..................................................................................................... 7 2.1 Culture of iPSC lines .............................................................................................................. 7 2.2 Editing of iPSC lines with CRISPR/Cas9 technology .................................................... 8 2.3 Single cells dilution: ................................................................................................................ 8 2.4 Genomic DNA extraction ...................................................................................................... 9 2.5 Screening of single-cell derived iPSC lines using PCR and Sanger sequencing ...... 9 2.6 Genome integrity and cells authentication analysis. .....................................................10 2.7 Quality assessment of iPSCs using Flow cytometry. .....................................................11 2.8 Quality assessment of iPSCs using Immunofluorescence staining (IF). ..................11 2.9 Assessment of differentiation potential of iPSC using embryoid body (EB) differentiation assay. ...................................................................................................................12 2.10 RNA isolation, cDNA synthesis for characterization of iPSCs using TaqMan hPSC Scorecard. ..........................................................................................................................12 2.11 Mutation analysis with quantitative PCR (qPCR). ....................................................12 2.12 POLR2A activity assay. .....................................................................................................13 2.13 Neural crest differentiation of iPSC ZEB2 knock-out line and its isogenic control. ..........................................................................................................................................................13 3 Results ............................................................................................................................... 15 3.1 Generation of iPSCs lines deficient for ZEB2, POLR2A, SCN1A and NCDN. .......15 3.2 Characterization of iPSC lines POLR2A K.D.2 and ZEB2 K.O.23. .........................17 3.2.1 Quality assessment using Flow cytometry and IF ......................................................18 3.2.2 Pluripotency and differentiation potential analysis ...................................................19 3.2.3 Mutation analysis using qPCR .......................................................................................20 3.3 POLR2A activity assay ........................................................................................................21 3.4 Neural crest cell (NCC) differentiation of ZEB2 K.O.23 iPSC line ..........................22 4. Discussion ........................................................................................................................ 23 Acknowledgments .............................................................................................................. 26 References ............................................................................................................................ 27 Abstract Induced pluripotent stem cells (iPSCs) can self-renew and differentiate into many other cell types. IPSCs are derived from somatic cells, and upon reprogramming, they share an expression profile similar to embryonic stem cells (ESCs). Among their many applications, iPSCs are an advantageous tool for disease modelling, offering an accurate system to study human molecular networks associated with specific phenotypes. Moreover, progress in genome editing technologies improved the possibilities for investigation of genotype-phenotype relationship for diseases characterized by defined genetic variants. Indeed, CRISPR/Cas9 edited iPSCs lines from healthy donors offer the possibility to investigate molecular networks with comparison to an isogenic control line. Furthermore, the ability of iPSCs to differentiate into neural cells, makes them a good model for studying neurodevelopmental diseases (NDDs). NDDs are characterized by heterogenous genetics and phenotypes. Heterozygous gene variants in the alpha 1 subunit of the sodium-voltage gated channel 1.1 (SCN1A) and in Neurochondrin (NCDN) have been associated with epilepsy. While many variants defining NDDs are associated with genes of transcriptional networks, e.g. the zinc-finger E-box binding homeobox 2 transcription factor (ZEB2) or the RPB1 subunit of RNA polymerase II complex (POLR2A). Although published animal model systems are available, there is a lack of human derived systems to investigate the gene function in disrupted molecular networks in NDDs. In this project, IPSCs deficient for SCN1A, NCDN, ZEB2 and POLR2A were generated using CRISPR/Cas9. To further evaluate the quality of the cell lines as iPSCs model, a POLR2A knock down (K.D.) line carrying a 4 bp insertion and a ZEB2 knock out (K.O.) line carrying a 790 deletion were characterized. Pluripotency and differentiation potential were confirmed by flow cytometry analysis, immunostaining, and qPCR. Both lines maintained genome integrity and editing in the top predicted off targets was excluded with PCR and Sanger sequencing screening. Furthermore, ZEB2 is involved in induction of neural crest cells (NCC); ZEB2 deficient line and the control behave similarly after a week of NCC differentiation. In contrast, POLR2A variants suggest slowing of transcription compared to the wild-type, therefore rate of transcription was measured performing an activity assay. No relevant differences between POLR2A K.D. and control line were observed in transcription rate of early pre-mRNA. List of abbreviations ASD Autism spectrum disorder BMP Bone morphogenetic protein bp Base pairs cDNA Complementary DNA chr chromosome DNA Deoxyribonucleic acid DRB 5,6-Dichloro-1-beta-Ribo-furanosyl Benzimidazole DS Dravet Syndrome EB Embryoid body ESCs Embryonic stem cells FACS Fluorescent activated cells sorting GFP Green fluorescence protein gRNA guide-RNA hPSCs Human pluripotent stem cells ID Intellectual disability IF Immunofluorescence staining iPSCs Induced pluripotent stem cells K.D. Knock down K.O. Knock out LN-521 Laminin-521 NCC Neural crest cells NDD Neurodevelopmental disease/disorder NSC Neural stem cells PCR Polymerase chain reaction qPCR Quantitative PCR Rhoki Rho kinase inhibitor RNA Ribonucleic acid SNP Single nucleotide polymorphism wp well cells culture plate WT Wild Type 1 Introduction 1.1 Characteristics and applications of Induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells (iPSCs) are characterized by the ability to self-renew and the capacity to differentiate into a plethora of other cell types. They are derived from somatic cells by forced expression of a set of transcription factors to become pluripotent, a process termed reprogramming. Different approaches have been developed for cell reprogramming, to introduce a set of gene encoding transcription factors into the cells. Expression of these so-called reprogramming factors triggers a change in gene expression leading to an expression profile similar to embryonic stem cells (ESCs) (1). Established protocols use vectors carrying transcription factors OCT4, SOX2, KLF4, and MYC. Additional factors have been described. The first two factors (i.e. OCT4 and SOX2) promote expression of pluripotency genes and repress expression of differentiation-directing genes. The second two (i.e. KLF4 and MYC) participate in altering chromatin structure allowing OCT4 and SOX2 to bind to DNA (2). Moreover, the preferred technologies for introduction of reprograming factors into cells are transfection (non-viral) methods or transduction by non-integrating viruses, such as Sendai virus, respectively, to avoid possible integration of viral DNA into the cell’s genomic DNA (1). IPSCs are an important tool for research owing to their multiple possible applications in different research fields. One example is the use of iPSCs as disease-modelling systems. As they possess the capacity to give rise to various types of differentiated cells, they enable the investigation of specific questions on
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