Direct conversion of somatic cells utilizing CRISPR/ Direct conversion of somatic cells utilizing...

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Transcript of Direct conversion of somatic cells utilizing CRISPR/ Direct conversion of somatic cells utilizing...

  • Helmholtz Zentrum München, Institut für Entwicklungsgenetik

    Direct conversion of somatic cells utilizing CRISPR/Cas9

    Benedict Samuel Rauser

    Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für

    Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung

    des akademischen Grades eines

    Doktors der Naturwissenschaften

    genehmigten Dissertation.

    Vorsitzender: Prof. Dr. Aphrodite Kapurniotu

    Prüfer der Dissertation: 1. Prof. Dr. Wolfgang Wurst

    2. Prof. Angelika Schnieke, Ph.D.

    Die Dissertation wurde am 20.06.2017 bei der Technischen Universität München

    Eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,

    Landnutzung und Umwelt am 04.12.2017 angenommen.

  • II

    INDEX

    1 Abstract .......................................................................................................................... 2

    2 Zusammenfassung ......................................................................................................... 3

    3 Introduction .................................................................................................................... 6

    3.1 Midbrain dopaminergic neurons and cell replacement strategies ............................ 6

    3.1.1 Midbrain dopaminergic neurons and Parkinson’s disease ................................... 6

    3.1.2 Different routes for cell replacement strategies in PD .......................................... 7

    3.1.2.1 Transcription factor combinations utilized for the direct conversion of

    fibroblasts to DA neurons .............................................................................. 9

    3.1.2.2 Roles of Ascl1, Lmx1a, Nurr1 in mdDA development and direct conversion of

    fibroblasts to DA neurons .............................................................................10

    3.2 The CRISPR/Cas9 system and its implications for genome editing and

    transcriptional regulation .......................................................................................12

    3.2.1 CRISPR/Cas9 - an adaptive immune system in bacteria and archaea ...............12

    3.2.2 Repurposing CRISPR/Cas9 technology for genome editing ...............................14

    3.2.3 Transcriptional modification by CRISPR/Cas9 ...................................................15

    4 Aim of the thesis ............................................................................................................20

    5 Results ..........................................................................................................................22

    5.1 Exogenous gene expression strategies for direct conversion of MEFs to DA

    neurons .................................................................................................................22

    5.1.1 Limitations of direct reprogramming utilizing multiple viruses for gene delivery ..22

    5.1.2 Limitations of multi-cistronic constructs for direct reprogramming ......................24

    5.1.3 Inefficient ribosome skipping at 2A sites results in fusion proteins .....................25

    5.1.4 The reprogramming efficiency of ALN is independent of doxycycline

    concentrations ...................................................................................................27

    5.1.5 Ribosome skipping at 2A sites is not affected by expression levels ...................29

    5.1.6 Successful generation of PITX3+ DA neurons by forskolin treatment .................30

    5.2 Induction of endogenous genes and direct reprogramming of astrocytes to neurons

    utilizing the CRISPR/Cas9 technology ...................................................................33

  • III

    5.2.1 Design of gRNAs and implementation of a screening assay to detect induction of

    endogenous genes ............................................................................................33

    5.2.2 Screening for dCas9 fusion proteins to induce endogenous Ascl1 .....................35

    5.2.2.1 The SpyTag system improves endogenous gene induction ..........................35

    5.2.2.2 Requirement of a new screening platform for SAM and VPR systems .........38

    5.2.2.3 Increasing the number of gRNAs has a synergistic effect on gene induction 40

    5.2.2.4 SAM and VPR systems are superior to SpyTag for Ascl1 activation .............42

    5.2.2.5 Combining SAM and VPR systems has a synergistic effect on Ascl1

    expression ....................................................................................................44

    5.2.3 Design and generation of lentiviral vectors for SAM and dCas9-VPR delivery ...47

    5.2.4 Challenges in lentiviral Cas9 packaging .............................................................50

    5.2.5 Direct reprogramming of astrocytes to neurons utilizing SAM and VPR .............53

    6 Discussion .....................................................................................................................58

    6.1 Limitations of exogenous gene expression for direct reprogramming ....................58

    6.1.1 The requirement of co-transductions limits the reprogramming efficiency ..........58

    6.1.2 The high reprogramming efficiency of Caiazzo et al., seems to be influenced by

    the reporter system ............................................................................................58

    6.1.3 Forskolin treatment enables the generation of PITX3+ DA neurons ....................59

    6.1.4 Inefficient ribosome skipping at 2A sites results in fusion proteins and cell death

    ..........................................................................................................................61

    6.2 Utilizing CRISPR/Cas9 technology for gene induction and direct cell conversion ..62

    6.2.1 The importance of gRNA screenings for transcriptional activation ......................62

    6.2.2 Screenings of dCas9 fusion proteins for an efficient gene induction ...................63

    6.2.3 Synergistic activating effect of SAM and VPR systems ......................................65

    6.2.4 Challenges in lentiviral delivery of dCas9 systems .............................................67

    6.2.5 Direct conversion of astrocytes to neurons utilizing VPR and SAM ....................68

    7 Conclusion and future perspectives ...............................................................................71

    8 Material and methods ....................................................................................................73

    8.1 Material .................................................................................................................73

    8.2 Methods ................................................................................................................85

  • IV

    8.2.1 Isolation and culture of primary cells and established cell lines ..........................85

    8.2.1.1 Storage and culture of stable cell lines .........................................................85

    8.2.1.2 Isolation and culture of primary fibroblasts ...................................................85

    8.2.1.3 Isolation and culture of primary astrocytes ...................................................86

    8.2.1.4 Coating of cover slips for cell attachment .....................................................86

    8.2.1.5 Lipofection ....................................................................................................86

    8.2.2 Preparation of lentiviruses and titer determination ..............................................87

    8.2.3 Direct reprogramming of somatic cells ...............................................................89

    8.2.3.1 Reprogramming of MEFs .............................................................................89

    8.2.3.2 Reprogramming of astrocytes ......................................................................89

    8.2.4 Immunocytochemistry and microscopy ..............................................................90

    8.2.5 Luciferase assay analysis ..................................................................................90

    8.2.6 RT-qPCR ...........................................................................................................91

    8.2.7 Western blot .......................................................................................................92

    8.2.8 Isolation of nucleic acids ....................................................................................93

    8.2.8.1 Isolation of RNA ...........................................................................................93

    8.2.8.2 Purification of DNA .......................................................................................93

    8.2.8.3 Agarose gel electrophoresis .........................................................................93

    8.2.9 DNA plasmid preparations .................................................................................93

    8.2.10 Cloning of new constructs ..................................................................................94

    8.2.10.1 Polymerase chain reaction ...........................................................................94

    8.2