Verfügbarkeit: Novel systems and delivery methods for enhanced safety in human genome editing

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Bibliographische Detailangaben
Beteilige Person:	Müller, Maximilian (VerfasserIn)
Format:	Hochschulschrift/Dissertation Buch
Sprache:	Englisch
Veröffentlicht:	Freiburg im Breisgau März 2018
Schlagwörter:	Hochschulschrift
Links:	http://d-nb.info/1170196535/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030742041&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Umfang:	IV, 154 Seiten Illustrationen, Diagramme 30 cm

Internformat

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Datensatz im Suchindex

DE-BY-UBR_call_number	24/18.0877
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adam_text	1. IN TR O D U C TIO N ...................................................................................................................................................................................1 1.1 G ENOM E E D IT IN G ...............................................................................................................................................................................1 1.2 P ROGRAMMABLE N UCLEASES ........................................................................................ 2 1.2.1 MEGANUCLEASES ................................................................................................................................... 2 1.2.2 ZINC-FINGER NUCLEASES ......................................................................................................................... 3 1.2.3 TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES ................................................................................ 4 1.2.4 RNA-GUIDED NUCLEASES ........................................................................................................................ 6 1.3 R EPAIR M ECHANISMS OF DNA D OUBLE -S TRAND B REAKS ............................................................................................................9 1.3.1 HOMOLOGY DIRECTED REPAIR ....................................................................................................... ........ 10 1.3.2 NON-HOMOLOGOUS END-JOINING ......................................................................................................... 11 1.4 T HERAPEUTIC G ENOME EDITING W ITH DESIGNER NUCLEASES ......................................................................................................13 1.5 O FF - T A R G E TIN G ...............................................................................................................................................................................15 1.5.1 IDENTIFICATION O F NUCLEASE OFF-TARGET SITES ...................................................................................... 15 1.5.2 M ITIGATING OFF-TARGET A C TIV ITY ......................................................................................................... 24 1.6 D ELIVERY OF DESIGNER NUCLEASES ................................................................................................................................................ 30 1.6.1 VIRAL DELIVERY METHODS .................................................................................................................... 30 1.6.2 NON-VIRAL DELIVERY M ETHODS ............................................................................................................ 33 1.7 A IM OF THE T HESIS ......................................................................................................................................................................... 36 2. MATERIAL A N D M E T H O D S ......................................................................................................................................................... 37 2.1 S TANDARD M OLECULAR BIOLOGY METHODS USED THROUGHOUT THIS THESIS ..........................................................................37 2.1.1 RESTRICTION DIGEST ............................................................................................................................ 37 2.1.2 LIGATION ............................................................................................................................................. 37 2.1.3 GIBSON ASSEMBLY ............................................................................................................................ 37 2.1.4 STANDARD PCR AM PLIFICATION ............................................................................................................ 37 2.1.5 TRANSFORMATION O F DNA INTO COMPETENT E. COU. ............................................................................... 37 2.1.6 DNA EXTRACTION FRO M BACTERIA (MINI, M IDI PREPARATIONS O F DNA) .................................................. 38 2.1.7 AGAROSE GEL ELECTROPHORESIS ........................................................................................................... 38 2.1.8 DNA P URIFICATION ............................................................................................................................. 38 2.2 M ETHODS USED IN S T C AS 9 PROJECT ............................................................................................................................................ 39 2.2.1 CAS 9 EXPRESSION PLASMIDS .............................................................................................................. 39 2.2.2 SINGLE GUIDE RNA EXPRESSION PLASM IDS ........................................................................................... 39 2.2.3 PLASMIDS FO R EPISOMAL GENE DISRUPTION ASSAY ................................................................................ 40 2.2.4 ENDOGENOUS GENE DISRUPTION ASSAYS ............................................................................................... 40 2.2.5 T7 ENDONUCLEASE I ASSAY ................................................................................................................. 41 2.2.6 OFF-TARGET SITE IDENTIFICATION AND DEEP SEQUENCING ......................................................................... 43 2.2.7 DEEP SEQUENCING TO QUANTIFY RGN ACTIVITY AT GENOMIC LOCI ............................................................ 43 2.2.8 PREPARING WHOLE CELL LYSATES FRO M CELL CULTURE ......................................... ...................................... 44 2.2.9 DETERMINATION O F PROTEIN CONCENTRATIONS USING THE DC PROTEIN ASSAY ............................................ 44 I 2.2.10 SDS-PAGE .....................................................................................................................................................................4 5 2.2.11 CELL CULTURE MODELS .................................................................................................................................................. 46 2.2.12 CELL CULTURING ............................................................................................................................................................... 46 2.2.13 CELL TRANSFECTIONS ...................................................................................................................................................... 46 2.2.14 FLOW CYTOMETRY ANALYSIS ......................................................................................................................................... 47 2.3 M ETHODS USED IN THE S NEAKY N UCLEASES PROJECT .................................................................................................................. 48 2.3.1 SNEAKY TALEN EXPRESSION PLASMIDS ......................................................................................................................... 48 2.3.2 SNEAKY CAS9 EXPRESSION PLASMIDS ............................................................................................................................ 49 2.3.3 CLONING O F THE LENTIVIRAL PLASMID FO R CCR5 EXPRESSION ..................................................................................... 49 2.3.4 IN VITRO TRANSLATION ......................................................................................................................................................... 50 2.3.5 IN VITRO CLEAVAGE ............................................................................................................................................................. 50 2.3.6 IN VITRO TRANSCRIPTION O F SINGLE GUIDE RNA (GRNA) ............................................................................................. 51 2.3.7 EXPRESSION O F SNEAKY TALENS .................................................................................................................................... 53 2.3.8 PURIFICATION O F SNEAKY TALENS ................................................................................................................................... 53 2.3.9 EXPRESSION O F SNEAKY CAS9 AND ................................................................................................................................. 54 2.3.10 PURIFICATION O F SNEAKY CAS9 ................................................................................................................................. 54 2.3.11 EXPRESSION O F SNEAKY AUREIN-CAS9 ........................................................................................................... 56 2.3.12 PURIFICATION AUF SNEAKY AUREIN-CAS9 ................................................................................................................. 56 2.3.13 COOMASSIE STAINING ..................................................................................................................................................5 7 2.3.14 CELL CULTURE MODELS ..................................................................................................................................................5 7 2.3.15 CULTURING O F ADHERENT CELL LINES ............................................................................................................................ 58 2.3.16 CELL COUNTING AND VIABILITY ASSESSMENT ............................................................................................................ 58 2.3.17 TRANSFECTIONS OFHEK293T CELLS AND CHO CELLS .............................................................................................. 59 2.3.18 NUCLEOFECTION OFHEK293T CELLS .......................................................................................................................... 60 2.3.19 GENERATION O F A CELL LINE CONSTITUTIVELY EXPRESSING CCR5 .......................................................................... 61 2.3.20 INCUBATION O F CELLS WITH SNEAKY NUCLEASE PROTEIN ........................................................................................ 62 2.3.21 IMMUNOFLUORESCENCE MICROSCOPY ...................................................................................................................... 63 2.3.22 T7 ENDONUCLEASE I ASSAY ........................................................................................................................................ 64 CHARACTERIZATION OF THE STREPTOCOCCUS THERMOPHILUS CRISPR/CAS9 SYSTEM FOR SPECIFIC EDITING OF THE H U M A N G E N O M E ......................................................................................................................................................................6 6 3. PROJECT IN T R O D U C T IO N ............................................................................................................................................................ 6 6 4 . RESULTS...............................................................................................................................................................................................6 8 4 .1 E XPRESSION LEVELS OF C AS 9 ORTHOLOGUES .............................................................................................................................. 68 4 .2 A CTIVITY OF S T CRISPR-C AS 9 SYSTEMS IN H U M A N CELLS .........................................................................................................68 4.2.1 EPISOMAL DSEGFP REPORTER ASSAY .............................. 68 4.2.2 CYTOTOXICITY OF ST CRISPR-CAS9 SYSTEMS IN HUMAN CELLS ................................................................................. 71 4.2.3 COMPARISON O F RGN CLEAVAGE ACTIVITIES AT ENDOGENOUS HUMAN LO C I ........................................................ 73 4.5 S PECIFICITY OF S TREPTOCOCCUS THERMOPHILUS CRISPR-C AS 9 SYSTEMS ............................................................................... 77 5. DISC U SSIO N ...................................................................................................................................................................................... 79 5.1 A CTIVITY OF S T - BASED RGN S ....................................................................................................................................................... 8 0 5.2 S PECIFICITY OF S T - BASED RGN S .................................................................................................................................................. 81 SNEAKY NUCLEASES FOR CELL-SPECIFIC DELIVERY OF TALENS A N D CASS ....................................................................... 8 3 6. PROJECT IN T R O D U C TIO N ............................................................................................................................................................83 6.1 T HE SNEAKING LIGAND TECHNOLOG Y .......................................................................................................................................... 84 6.2 S NEAKY D ESIGNER N UCLEASES ...................................................................................................................................................... 85 6.2.1 SNEAKY TALENS ................................................................................................................................ 86 6.2.2 SNEAKY CAS9 ...................................................................................................................................... 87 7. RESULTS..............................................................................................................................................................................................8 9 7 .1 S NEAKY TALEN FUSION PRO TEIN S ................................................................................................................................................89 7.1.1 SNEAKY TALENS ARE ACTIVE IN VITRO .................................................................................................... 89 7.1.2 SNEAKY TALENS ARE ACTIVE IN HUMAN CEILS. ....................................................................................... 90 7.1.3 CREATION O F A CELL LINE CONSTITUTIVELY EXPRESSING CCR5 .................................................................... 90 7.1.4 SNEAKY TALENS CAN BE DELIVERED INTO TARGET CELLS ............................................................................ 91 7.1.5 ASSESSMENT O F SNEAKY TALENS A CTIVITY ............................................................................................ 94 7.1.6 EXPRESSION AND PURIFICATION ............................................................................................................. 94 7.2 S NEAKY C AS 9 FUSION PRO TEINS ....................................................................................................................................................98 7.2.1 EXPRESSION AND PURIFICATION ........................................................................ 98 7.2.2 SNEAKY CAS9 IS ACTIVE IN VITRO ......................................................................................................... 101 7.2.3 SNEAKY CAS9 IS ACTIVE IN HUMAN CELLS ............................................................................................. 103 7.2.4 ASSESSMENT O F CELL-SPECIFIC DELIVERY O F SNEAKY CAS9 ...................................................................... 104 7.3 S NEAKY A UREIN -C AS 9 ................................................................................................................................................................. 106 7.3.1 EXPRESSION AND PURIFICATION ............................................................................................................ 106 7.3.2 CELL-SPECIFIC ACTIVITY O F SNEAKY AUREIN CAS9 AT ENDOGENOUS COSMC ............................................ 109 8. DISCU SSIO N................................................................................................................................................................................... 112 8.1 P ROTEIN EXPRESSION AND PURIFICATION ................................................................................................................................... 113 8.2 S NEAKY NUCLEASES RETAIN THEIR DNA- CLEAVAGE A C TIV IT Y ...................................................................................................114 8.3 C ELL SPECIFIC DELIVERY AND CLEAVAGE ACTIVITY .......................................................................................................................115 8 .4 C O N C L U S IO N ................................................................................................................................................................................ 121 9. REFERENCES................................................................................................................................................................................... 122 10. A P P E N D IX ....................................................................................................................................................................................... 141 10.1 A M IN O ACID SEQUENCES OF C AS 9 PROTEINS USED IN THIS THESIS ................................................................................... 141 10.2 DNA SEQUENCES OF GRNAS USED IN THIS STUDY............................................................................................................. 143 III 10.3 A M IN O ACID SEQUENCES OF TALEN PROTEINS AND FUSION D O M AINS USED IN THIS THESIS .......................................... 144 10.4 S EQUENCING RESULTS FOR PRKDC- SPECIFIC RGN S ....................................................................................... 147 10.5 S EQUENCING RESULTS FOR CARD11- SPECIFIC RGN S .................................................................................... 148 10.6 P LASMIDS USED IN THIS TH E S IS .............................................................................................................................................. 149 10.7 O LIGONUCLEOTIDES USED IN THIS THESIS .............................................................................................................................. 150
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spellingShingle	Müller, Maximilian Novel systems and delivery methods for enhanced safety in human genome editing
subject_GND	(DE-588)4113937-9
title	Novel systems and delivery methods for enhanced safety in human genome editing
title_auth	Novel systems and delivery methods for enhanced safety in human genome editing
title_exact_search	Novel systems and delivery methods for enhanced safety in human genome editing
title_full	Novel systems and delivery methods for enhanced safety in human genome editing vorgelegt von Maximilian Müller
title_fullStr	Novel systems and delivery methods for enhanced safety in human genome editing vorgelegt von Maximilian Müller
title_full_unstemmed	Novel systems and delivery methods for enhanced safety in human genome editing vorgelegt von Maximilian Müller
title_short	Novel systems and delivery methods for enhanced safety in human genome editing
title_sort	novel systems and delivery methods for enhanced safety in human genome editing
topic_facet	Hochschulschrift
url	http://d-nb.info/1170196535/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030742041&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT mullermaximilian novelsystemsanddeliverymethodsforenhancedsafetyinhumangenomeediting

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