CRISPR/Cas9-mediated genome engineering of the SMARCB1 locus

Fachbereich Biologie der Technischen Universität Darmstadt

Zur Erlangung des Grades Doctor rerum naturalium (Dr. rer. nat.)

Dissertation von Marion Brigitte Lechler

Erstgutachterin: Prof. Dr. Ulrike A. Nuber Zweitgutachterin: Prof. Dr. M. Cristina Cardoso

Darmstadt 2020

Lechler, Marion Brigitte: CRISPR/Cas9-mediated genome engineering of the SMARCB1 gene locus, Stem Cell and Developmental Biology Darmstadt, Technische Universität Darmstadt, Jahr der Veröffentlichung der Dissertation auf TUprints: 2021 URN: urn:nbn:de:tuda-tuprints-118851 Tag der mündlichen Prüfung: 10. September 2020

Veröffentlicht unter CC-BY-SA 4.0 International https://creativecommons.org/licenses/

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I. Table of contents

I...... Table of contents I II. .... Abbreviations IV III. .. Abstract / Zusammenfassung VII 1...... Introduction 1 1.1. The tumour suppressor gene SMARCB1 1 1.2. BRG1-associated factor (BAF) complexes 2 1.3. Diseases associated with mutations affecting the SMARCB1 gene 7 1.4. CRISPR/Cas9-mediated genome engineering 14 1.5. Aim of this thesis 17 2...... Materials and methods 22 2.1. Molecular biology methods 22 2.1.1. Polymerase chain reaction (PCR) amplification of DNA 22 2.1.2. Purification of DNA samples 23 2.1.3. Restriction endonuclease digestion 24 2.1.4. Agarose gel electrophoresis 25 2.1.5. Blunting of cohesive DNA ends 27 2.1.6. Dephosphorylation of DNA ends 27 2.1.7. Ligation of DNA fragments 28 2.1.8. Generation of chemically-competent Escherichia coli 29 2.1.9. Transformation of Escherichia coli DH5α and Stbl3 31 2.1.10. Colony PCR 32 2.1.11. Plasmid DNA preparations 33 2.1.12. Sanger sequencing of DNA samples 36 2.1.13. TA-cloning 36 2.1.14. Overlap extension method 38 2.1.15. Cloning of guide RNA sequences into multiplex CRISPR/Cas9(n) plasmids 39 2.1.16. Gateway cloning 41 2.1.17. RNA isolation 42 2.1.18. cDNA synthesis 43 2.1.19. Quantitative reverse transcription PCR (RT-qPCR) 44 2.2. Biochemical methods 46 2.2.1. sample preparation and protein concentration measurement 46 2.2.2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis 47 2.2.3. Immunoblotting 50 2.3. Cell culture methods 53 2.3.1. Cultivation and transfection of the human embryonic kidney cell line HEK293T 53 2.3.2. Cultivation and transfection of human induced pluripotent stem cells (hiPSC) 54 2.3.3. Cultivation and transfection of the human breast epithelial cell line MCF10A 55 2.3.4. Cultivation of mouse neural stem and progenitor cells as neurospheres (NSPs) 56 2.3.5. Flow Cytometry analysis and sort (FACS) 57 2.3.6. Electroporation of human cell lines 58 2.3.7. Generation and concentration of lentiviral particles 61 2.3.8. Transduction of mammalian cell lines 62

I Table of contents I

3...... Results 63 3.1. Design and generation of constructs for CRISPR/Cas9-mediated genome engineering of the SMARCB1 locus in human cells 63 3.1.1. Design and generation of constructs for CRISPR/Cas9-mediated genome engineering of human cell lines with AT/RT-associated SMARCB1 mutations 65 3.1.2. Design and generation of constructs for CRISPR/Cas9-mediated genome engineering of human cell lines with CSS-associated SMARCB1 mutations 69 3.1.3. Design and generation of gRNA and Cas9 expression plasmids for CRISPR/Cas9-mediated genome engineering of the SMARCB1 locus in human cells 70 3.2. Establishment and optimisation of CRISPR/Cas9-mediated genome engineering methods in HEK293T cells 74 3.2.1. Establishment and optimisation of the CRISPR/Cas9-mediated genome engineering of the SMARCB1 locus in HEK293T cells using the generated AT/RT-associated constructs 74 3.2.2. Establishment and optimisation of the CRISPR/Cas9-mediated genome engineering of the SMARCB1 locus in HEK293T cells using the generated CSS-associated constructs 79 3.3. Establishment and optimisation of methods for CRISPR/Cas9-mediated genome engineering in hiPSCs 80 3.4. Establishment and optimisation of methods for CRISPR/Cas9-mediated genome engineering in MCF10A cells 84 3.5. Loss-of-function mutations in SMARCB1 de-regulate expression of Hes in murine neural stem and in HEK293T cells 86 3.5.1. Loss-of-function mutations in SMARCB1 de-regulate expression of Hes genes in E14.5 mouse brain samples and in HEK293T cells 86 3.5.2. Cloning and lentiviral packaging of murine Hes5-IRES-mKate2 overexpression and IRES- mKate2 control plasmids for the transduction of murine neural stem cells 89 3.5.3. Establishment and optimisation of methods to overexpress Hes5 in murine NSCs 90 4...... Discussion 93 4.1. Establishment and optimisation of methods and constructs for CRISPR/Cas9-mediated genome engineering of the SMARCB1 gene locus in human cell lines 93 4.1.1. Transfer of large constructs into hiPSC#33 and re-evaluation of the HDR constructs 94 4.1.2. Transfer of large constructs into MCF10A cells 97 4.2. The designed multi-colour strategies for the CRISPR/Cas9-mediated genome engineering in comparison to other cellular modelling approaches at the SMARCB1 locus 100 4.2.1. The designed three-colour reporter strategy in comparison to another cellular AT/RT model 100 4.2.2. The designed two-colour strategy in comparison to another cellular CSS model 102 4.3. Loss-of-function mutations in SMARCB1 de-regulate expression of Hes genes 104 5...... Outlook 106 6...... Bibliography 107

I Table of contents II

7...... Appendix 117 7.1. Materials 117 7.1.1. List of oligonucleotides 117 7.1.2. List of restriction endonucleases 120 7.1.3. Guide RNA positioning within the genomic sequence of SMARCB1 121 7.1.4. Graphical overview of all constructs generated for the CRISPR/Cas9-mediated genome engineering of the human SMARCB1 locus 122 7.1.5. Graphical overview of the cloning of the original lentiviral Hes5-IRES-mKate2 plasmid for overexpression of Hes5 128 7.1.6. Information of plasmids generated or used in this thesis 128 7.2. Publications 134 7.3. Scholarship 134 7.4. Contributions of others 134 7.5. Curriculum vitae 135 IV. ... Acknowledgements IX V. .... Assertion – Ehrenwörtliche Erklärung X

I Table of contents III

II. Abbreviations ABBREVIATION DESCRIPTION 1° Primary, e.g. 1° antibody = primary antibody 2° Secondary, e.g. 2° antibody = secondary antibody ° C Degree Celsius µL Microliter A260/A280 Ratio of the absorption at 260 and 280 nm, value for the purity of nucleic acids AMP Adenosine monophosphate approx. Approximately AT/RT Atypical-teratoid/rhabdoid tumour ATP Adenosine triphosphate BAF BRG1- or BRM-associated factor bFGF Basic fibroblast growth factor bp BSA Bovine serum albumin BSD Blasticidin S deaminase gene Cas9n Cas9 nickase cDNA Complementary DNA CRISPR Clustered regularly interspaced palindromic repeat CSS Coffin-Siris syndrome

cT Threshold cycle (RT-qPCR)

∆cT Difference of cT values of the gene of interest and the housekeeping gene (RT-qPCR)

∆∆ cT Difference of delta cT values of treatment and control samples Da Dalton, unified atomic mass unit DH5α Laboratory strain of E. coli DMEM high glucose Dulbecco’s Modified Eagle's medium supplemented with 4500 mg/L glucose DMEM/F12 Dulbecco’s Modified Eagle's medium nutrient mixture F-12 DNA Deoxyribonucleic acid DNaseI Deoxyribonuclease I dNTP Deoxyribonucleotide triphosphate DPBS Dulbecco’s phosphate buffered saline E Embryonic day E. coli Escherichia coli EGF Epidermal growth factor eGFP::ZeoR Enhanced green fluorescent protein (eGFP) and zeocin resistance (ZeoR) fusion gene FCS Fetal calf serum g Gram gDNA Genomic DNA gRNA (single) guide RNA GSG Glycine-Serine-Glycine linker

H2Odest. Deionised water HDR Homology directed repair HEK293T Human embryonic kidney 293 cells expressing mutant SV40 antigen T Hes/HES Mammalian homologues of Drosophila hairy and Enhancer of split hiPSC(s) Human induced pluripotent stem cell(s) hiPSC#33 Human induced pluripotent stem cells cl