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Advanced Genome Engineering Using CRISPR/Cas9 and Transposases

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Advanced Genome Engineering Using CRISPR/Cas9 and Transposases Advanced Genome Engineering Using CRISPR/Cas9 and Transposases Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 14 der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Adrian Kovač aus Frankfurt am Main Frankfurt, 2020 Vom Fachbereich 14 der Johann Wolfgang Goethe – Universität als Dissertation angenommen. Dekan: Prof. Dr. Clemens Glaubitz Gutachter: Prof. Dr. Rolf Marschalek Datum der Disputation: 22.10.2020 Table of Contents 1 Introduction ......................................................................................................................... 1 1.1 Overview over gene therapy history and technology .................................................. 1 1.2 Integrating vectors ....................................................................................................... 2 1.2.1 Viral vectors ......................................................................................................... 2 1.2.2 Transposable elements ......................................................................................... 3 1.2.3 Transposon vectors ............................................................................................... 7 1.3 The SB transposon ..................................................................................................... 10 1.3.1 Sleeping Beauty transposon and transposase structure ...................................... 10 1.3.2 Sleeping Beauty transposition mechanism and target selection ......................... 12 1.3.3 Sleeping Beauty optimization and variants ........................................................ 15 1.3.4 Applications of the Sleeping Beauty system ...................................................... 17 1.4 Site-specific genome engineering .............................................................................. 19 1.4.1 Nuclease-based genome engineering ................................................................. 19 1.4.2 The CRISPR/Cas9 system .................................................................................. 21 1.4.3 Nuclease-free editing and other site-specific methods ....................................... 24 1.5 Natural and artificial targeted insertion systems ....................................................... 25 1.5.1 Natural targeted insertion systems ..................................................................... 27 1.5.2 Artificial retargeting of integrating vectors ........................................................ 30 1.5.3 Sleeping Beauty retargeting ................................................................................ 33 1.5.4 Targeting of ribosomal DNA ............................................................................. 35 2 Material and methods ........................................................................................................ 38 2.1 Material ...................................................................................................................... 38 2.1.1 Chemicals ........................................................................................................... 38 2.1.2 Media, buffers and solutions .............................................................................. 38 2.1.3 Kits ..................................................................................................................... 39 2.1.4 Other consumables ............................................................................................. 40 2.1.5 Equipment .......................................................................................................... 40 2.1.6 Antibodies and enzymes ..................................................................................... 40 2.1.7 Bacterial strains and eukaryotic cell lines .......................................................... 42 2.1.8 Plasmids ............................................................................................................. 42 2.1.9 Primers ............................................................................................................... 44 2.1.10 Other oligonucleotides ....................................................................................... 49 2.1.11 Software ............................................................................................................. 50 2.1.12 Services .............................................................................................................. 51 2.2 Methods ..................................................................................................................... 51 2.2.1 Plasmid construction .......................................................................................... 51 2.2.2 Cell culture and transfection .............................................................................. 54 2.2.3 Selection-based transposition assays .................................................................. 54 2.2.4 Western Blot ....................................................................................................... 54 2.2.5 Electrophoretic mobility shift assay (EMSA) .................................................... 55 2.2.6 Generation of integration libraries ..................................................................... 56 2.2.7 Integration site sequencing and analysis ............................................................ 57 2.2.8 PCR-based insertion site analysis ...................................................................... 57 2.2.9 In vitro digestion with Cas9 ............................................................................... 58 2.2.10 T7 Endonuclease assay ....................................................................................... 58 2.2.11 TIDE assay ......................................................................................................... 59 2.2.12 Sleeping Beauty mutagenesis ............................................................................. 59 2.2.13 Assembly of the SB mutant library .................................................................... 59 2.2.14 Screening of the SB mutant library .................................................................... 60 2.2.15 High-throughput FACS analysis ........................................................................ 61 2.2.16 Immunofluorescence microscopy ...................................................................... 61 3 Results ............................................................................................................................... 62 3.1 Retargeting of SB with dCas9 ................................................................................... 62 3.1.1 Generation of fusions between dCas9 and transposase components ................. 62 3.1.2 Transpositional activity of dCas9-SB100X ........................................................ 63 3.1.3 DNA-binding activities of dCas9 fusions .......................................................... 64 3.1.4 Integration library generation ............................................................................. 66 3.1.5 Validation of L1-directed sgRNAs .................................................................... 68 3.1.6 Validation of AluY-directed sgRNAs ................................................................ 69 3.1.7 AluY- and L1-targeted integration libraries ....................................................... 70 3.1.8 Validation of HPRT-directed sgRNAs ............................................................... 75 3.1.9 HPRT-targeted integration libraries ................................................................... 76 3.1.10 Design and validation of GSH-targeted sgRNAs ............................................... 77 3.1.11 GSH-targeted integration libraries ..................................................................... 78 3.1.12 Design and validation of TAn-targeted sgRNAs ................................................ 79 3.1.13 TAn -targeted integration libraries ...................................................................... 80 3.1.14 Generation of reduced-affinity SB mutants ....................................................... 82 3.1.15 Generation of a random SB mutant library ........................................................ 84 3.1.16 Screening of the mutant libraries ....................................................................... 85 3.1.17 Transposition with dCas9-SB(C42) ................................................................... 86 3.1.18 Targeting of single-copy loci with dCas9-SB(C42) ........................................... 87 3.1.19 Staged targeting with dCas9-SB100X and dCas9-SB(C42) .............................. 87 3.2 Retargeting of SB by ribosomal localization ............................................................. 88 3.2.1 Characterization of NoLS-SB100X fusions ....................................................... 88 3.2.2 Characterization of B23-SB100X ...................................................................... 89 3.2.3 B23-SB100X insertion libraries ......................................................................... 90 3.3 HDR enhancement with Cas9 fusions ....................................................................... 92 3.3.1 Characterization of Cas9-N57 and Cas9-N123 fusions ..................................... 92 3.3.2 Test of HDR enhancement using the TLR system ............................................
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