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CRISPR Interference to Model the Koolen-De Vries Syndrome in Ineurons Derived from Ipscs

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CRISPR Interference to Model the Koolen-De Vries Syndrome in Ineurons Derived from Ipscs CRISPR Interference to model the Koolen-de Vries Syndrome in iNeurons derived from iPSCs Bachelor Thesis Tessa van der Heijden June 2016 0 CRISPR Interference to model the Koolen- de Vries Syndrome in iNeurons derived from iPSCs Student Tessa van der Heijden [email protected] Student number 2064429 Supervisors Dr. Nael Nadif Kasri [email protected] Katrin Linda [email protected] Teacher Dr. Kees Rodenburg [email protected] Internship Department of Cognitive Neuroscience Radboudumc 6500 HB Nijmegen Donders Institute for Brain, Cognition and Behavior Centre for Neuroscience 6526 AJ Nijmegen Education Biology and Medical Laboratory Research Major Forensic Laboratory Research Academy for the Technology of Health and Environment Avans University of Applied Sciences Lovensdijkstraat 61-63 4818 AJ Breda Date Deadline Thesis: 13th of June 2016 Presentation: 27th of June 2016 Picture on front page derived from http://www.nature.com/news/crispr-1.17547 (consulted on 24-2-16) 1 Preface This thesis is the result of my graduation research project at the Radboud University Medical Center in Nijmegen, for my Bachelor program Biology and Medical Laboratory Research, affiliated to the academy for the technology of health and environment at Avans University of Applied Sciences, Breda. This project is conducted from September 2015 to June 2016. I would like to thank the principal investigator Dr. Nadif Kasri for offering me this research opportunity and for the supervision together with my supervisor Katrin Linda. I would also like to thank Dr. Kees Rodenburg for the supervision from school and my colleagues and fellow students for the great support and collaboration during my graduation internship. 2 Abstract The Koolen-de Vries Syndrome (KdVS) is a neurodevelopmental disorder, also called the 17q21.31 microdeletion syndrome, which is characterized by intellectual disability, developmental delay, epilepsy, hypotonia and distinct facial features.6 The main causative gene for this disease is the KANSL1 gene, which is part of the non-specific lethal (NSL), is involved in the acetylation of histone H4 lysine 16 (H4K16), an epigenetic process that is mainly important during development and mitosis.3,7 In order to study the pathogenesis of the KdVS, we used a cell line of induced pluripotent stem cells (iPSCs) wherein the KANSL1 gene was supposed to be knocked down by the sequence specific CRISPR interference (CRISPRi) technology. This technology is based on the co-expression of catalytically deficient endonuclease dead Cas9 protein (dCas9) and a sequence specific single guide RNA (sgRNA) that together form a complex, enabling sequence specific gene silencing of any gene complementary to the sgRNA.1 Transformation of the KANSL1 deficient cells into induced neurons (iNeurons) would allow us to investigate the pathogenesis of the disease during development. In order to integrate the dCas9 gene in iPSCs, we used two different constructs with an EF1alpha promoter and a doxycycline inducible promoter. However, lentiviral delivery of these constructs did not result in dCas9 expression. Now we have a CRISPRi WTC cell line of iPSCs (obtained from Mandegar et al. 2016) that enables dCas9 expression, which was proven by means of immunostainings. This allows us to test the sgRNAs for performing CRISPRi in iPSCs. 3 Samenvatting Het Koolen-de Vries Syndroom (KdVS) is een neuro-ontwikkelingsziekte, ook wel bekend als het 17q21.31 microdeletie syndroom, die wordt gekenmerkt door verstandelijke beperking, ontwikkelingsachterstanden, epilepsie, hypotonie en afwijkende gezichtskenmerken. Het belangrijkste gen dat verantwoordelijk is voor de ziekte is het KANSL1 gen dat deel uitmaakt van het non-specific lethal (NSL) complex en is betrokken bij de acetylatie in histoon H4 lysine 16 (H4K16). Dit is een belangrijk epigenetisch proces, vooral tijdens de ontwikkelingsfase en mitose van cellen.3,6 Het doel van het project was om de pathogenese van het KdVS te kunnen bestuderen door middel van het maken van een cellijn van induced pluripotent stem cells (iPSC’s). Daarin wordt het KANSL1 uitgeschakeld aan de hand van CRISPR interference (CRISPRi). Dit systeem is gebaseerd op de co- expressie van katalytisch deficiënt endonuclease dead Cas9 (dCas9) wat een complex vormt met het target specifieke single guide RNA (sgRNA) en zorgt voor het inactiveren van het target gen.1 Het idee is om deze KANSL1 deficiënte cellen te transformeren naar neuronen wat het analyseren van de pathogenese tijdens de ontwikkeling van neuronen mogelijk maakt. De iPSC’s werden getransduceerd met twee verschillende constructen om het dCas9 gen in het genoom van de cellen te integreren. Beide constructen bevatten een andere promotor, namelijk een EF1alfa promotor en een doxycycline induceerbare promotor. Echter heeft de lentivirale transductie van het dCas9-virus niet geleid tot de expressie van het dCas9 eiwit. Inmiddels maken we gebruik van een CRISPRi WTC cellijn van iPSC’s (afkomstig van Mandegar e.a., 2016) waarin het dCas9 eiwit wel tot expressie komt. Dit is bewezen middels immunocytochemie. Hierdoor kan het project voortgezet worden door verschillende sgRNA’s te testen om CRISPRi in iPSC’s te introduceren zodat het KANSL1 gen uitgeknokt wordt. 4 Contents Preface ..................................................................................................................................................... 2 Abstract ................................................................................................................................................... 3 Samenvatting ........................................................................................................................................... 4 1. Introduction ..................................................................................................................................... 6 2. Theoretical Background................................................................................................................... 7 ........................................................................................................................................................... 13 3. Materials and Methods ................................................................................................................. 14 Maintaining iPSCs .............................................................................................................................. 14 Making lentiviral constructs using calcium phosphate transfection ................................................. 14 Infection with lentiviral constructs.................................................................................................... 15 iNeuron differentiation ..................................................................................................................... 16 Preparation and western blot ........................................................................................................... 16 Immunofluorescence staining ........................................................................................................... 17 4. Results ........................................................................................................................................... 18 No KANSL1 knockdown observed in iPSCs transduced with construct 1 .......................................... 18 No dCas9 expression observed in iPSCs and iNeurons infected with constructs 1 and 2 ................. 19 Protein expression of dCas9 is present in CRISPRi WTC cell line ...................................................... 20 5. Discussion ...................................................................................................................................... 21 6. Conclusion ..................................................................................................................................... 23 7. Future perspective ........................................................................................................................ 23 8. References ..................................................................................................................................... 25 Supplementary data .............................................................................................................................. 27 Construct 1 ........................................................................................................................................ 27 Construct 2 ........................................................................................................................................ 28 Construct 3 ........................................................................................................................................ 29 Construct 4 ........................................................................................................................................ 30 5 1. Introduction In the department Cognitive Neuroscience at the University Medical Center Nijmegen (Radboudumc), there has been done research on cellular and molecular neurophysiology on several neurodevelopmental disorders. Cellular neurophysiological research has been done by means of electrophysiological recordings and micro-electrode arrays (MEAs) for analyzing cells at the single cell and network level. Furthermore, a nice in-vitro model of iNeurons derived from iPSCs is used to model e.g. the Kleefstra Syndrome for both
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