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Establishing CRISPR/Cas9-Mediated Functional Knock-Outs of Genes

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Establishing CRISPR/Cas9-Mediated Functional Knock-Outs of Genes Establishing CRISPR/Cas9-mediated functional knock-outs of genes TRNP1 and TMEM14B via microhomology-mediated end-joining DNA-repair in human induced pluripotent stem cells Master’s Thesis Submitted to the Institute of Molecular Biology University of Innsbruck in fulfillment of the requirements for the conferral of the degree of Master of Science (MSc) Author: Björn Felder, BSc Supervisor: Univ.-Prof. Frank Edenhofer, PhD Institute of Molecular Biology Department of Genomics, Stem Cell Biology and Regenerative Medicine LEOPOLD FRANZENS UNIVERSITÄT INNSBRUCK, AUSTRIA Co-Supervisor: Assoc.-Prof. Anna Falk, PhD Department of Neuroscience KAROLINSKA INSTITUTET STOCKHOLM, SWEDEN Innsbruck, October 2019 ii Contents Summary ............................................................................................................................ v 1. Introduction ................................................................................................................. 1 1.1. Genome engineering and the use of programmable nucleases ............................ 1 1.2. The CRISPR/Cas9 system .................................................................................... 2 1.3. Microhomology-mediated end-joining (MMEJ) DNA-repair ................................... 9 1.4. Induced pluripotent stem cells ............................................................................. 14 1.5. Aims of the Thesis ............................................................................................... 16 2. Materials and Methods ............................................................................................. 17 2.1. Materials .............................................................................................................. 17 2.2. Methods ............................................................................................................... 22 2.2.1. Molecular cloning and plasmid preparation .................................................. 22 2.2.2. gRNA and microhomologous sequences ..................................................... 22 2.2.3. Primer design ............................................................................................... 23 2.2.4. In vivo assembly and polymerase chain reaction for cloning ....................... 24 2.2.5. Restriction cloning and annealing of oligos .................................................. 25 2.2.6. Purification of PCR and restriction enzyme digest reactions ....................... 26 2.2.7. Ligation ......................................................................................................... 27 2.2.8. Agarose gel electrophoresis ......................................................................... 27 2.2.9. Transformation of competent cells ............................................................... 28 2.2.10. Bacterial culture and plasmid preparation ................................................ 29 2.2.11. Analysis of PCR-generated genomic and vector DNA ............................. 30 2.2.11.1. Isolation of genomic DNA ......................................................................... 30 2.2.11.2. Sequencing of vector products and PCR amplificates ............................. 30 2.2.12. Cell culture of human NHDF iPS cells ...................................................... 31 2.2.13. Cell imaging and fluorescence microscopy .............................................. 32 2.2.14. Generation and isolation of functional knock-out iPS cell lines ................ 32 2.2.15. Genotyping and screening for potential off-target cleavage ..................... 32 2.3. Lists of oligonucleotides and primers .................................................................. 34 2.4. List of plasmids .................................................................................................... 40 3. Results ....................................................................................................................... 41 3.1. Definition of gRNA target sites and microhomology sequences ......................... 41 3.1.1. In silico design of TRNP1 functional knock-out approach ............................ 41 3.1.2. In silico design of TMEM14B functional knock-out approach ....................... 42 3.2. Cloning modifications of pCRIS-PITCh expression vector system ...................... 44 3.3. Addition of empty gRNA cassette to px458-Cas9 expression vector .................. 45 iii 3.4. Introduction of gene specific gRNA sequences into px458_+PITCh gRNA vectors ............................................................................................................................ 48 3.5. Placement of transgenic expression construct under control of human EF1a promoter ......................................................................................................................... 50 3.6. Exchange of fluorescent reporter coding sequences, site-directed mutagenesis and poly-adenylation signal incorporation ...................................................................... 54 3.7. Introduction of gene specific microhomologous sequences into repair template vector ............................................................................................................................ 59 3.8. Cell maintenance of TRNP1 knock-out NHDF iPS cells and fluorescence imaging ............................................................................................................................ 64 3.9. Establishing monoclonal cell lines from single-cell sorted TRNP1-KO iPS cells . 67 3.10. PCR screen and genotyping of monoclonal TRNP1 functional knock-out iPS cell lines ......................................................................................................................... 68 3.11. Genomic off-target PCR screening and sequence analysis of TRNP1 target locus ......................................................................................................................... 74 4. Discussion ................................................................................................................ 76 4.1. To PITCh, or not to PITCh - the power lies in your hand .................................... 76 4.2. A convenient but effective screening method is key to the reliability of CRIS- PITCh experiments and gene manipulation ................................................................... 77 4.3. Physical prerequisites meet physiological limitations during CRISPR gene editing - a stem cell’s point of view ............................................................................................ 78 5. Outlook ...................................................................................................................... 81 6. List of Figures ........................................................................................................... 83 7. List of Tables ............................................................................................................ 85 8. References ................................................................................................................ 86 9. Supplementary figures and tables .......................................................................... 95 List of abbreviations ...................................................................................................... 115 iv Summary CRISPR/Cas9 genome editing is at the forefront of becoming a vital tool for the study of genes and their functions in live organisms and cells. One particularly promising method for the application of genome editing is to precisely integrate foreign DNA into target chromosomes, referred to as the PITCh knock-in system. The underlying mechanism within this gene manipulation method relies on an alternative DNA-repair pathway active in most dividing cells: microhomology-mediated end-joining (MMEJ) of broken DNA. This pathway has been harnessed to site-specifically knock-in foreign genes into the genome of host cells and animals with considerable precision and efficiency. During the course of this Master’s thesis, a functional knock-out approach using MMEJ- repair and the PITCh setup, to abolish the functions of human genes TRNP1 and TMEM14B in human induced pluripotent stem (iPS) cells, was pursued. These two genes are thought to play a major role during the development of the human cerebral neocortex, as they act and function specifically in basal radial glia (bRG) and intermediate progenitors (IPs) that reside in the outer subventricular zone (OSVZ) of the cortex. Their roles in genetic regulation and their effects in vivo have been modeled by overexpression and transcriptional repression studies (knock-down) in mouse brains. The factors’ roles in controlling the proliferation of progenitor cells in the OSZV as key regulators of cortex expansion and folding during development have been elucidated. However, a knock-out of these genes in the genome of human cells has thus far not been demonstrated. Therefore, this Thesis had the aim to perform such gene knock-outs in human iPS cell-derived cell models of the central nervous system. The materials and published guidelines of PITCh knock-in were adopted and modified for the purpose of achieving functional knock-outs of the genes through MMEJ integration of reporter genes at the coding gene loci. In silico design and molecular cloning of CRISPR donor constructs, including guide RNAs for precise locus targeting were the major tasks. Generated plasmids were introduced
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