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Generating Meganucleases with New Specificities by Directed Evolution Generating meganucleases with new specificities by directed evolution and rational engineering approaches Inauguraldissertation zur Erlangung des Grades Doktor der Naturwissenschaften Dr. rer. nat. Im Fachbereich Biologie und Chemie der Justus-Liebig-Universität Gießen vorgelegt von Diplom-Biologin Ines Fonfara Gießen im August, 2011 Die vorliegende Arbeit wurde im Rahmen des Graduiertenkollegs „Enzymes and Multienzyme Complexes Acting on Nucleic Acids“ (GRK 1384) und mit Finanzierung durch das DFG geförderte Schwerpunktprogramm (SPP 1170) „Directed Evolution to Optimize and Understand Molecular Biocatalysts“ am Institut für Biochemie des Fachbereich 08 der Justus-Liebig-Universität Gießen in der Zeit von April 2008 bis Juli 2011 unter Leitung von Prof. Dr. A. Pingoud durchgeführt. Erstgutachter: Prof. Dr. Alfred Pingoud Institut für Biochemie Fachbereich Biologie und Chemie Heinrich-Buff-Ring 58 35392 Gießen Zweitgutachter: Prof. Dr. Michael U. Martin Professur für Immunologie Fachbereich Biologie und Chemie Winchesterstrasse 2 35394 Gießen 2 Erklärung Ich erkläre: Ich habe die vorgelegte Dissertation selbständig und ohne unerlaubte fremde Hilfe und nur mit den Hilfen angefertigt, die ich in der Dissertation angegeben habe. Alle Textstellen, die wörtlich oder sinngemäß aus veröffentlichten Schriften entnommen sind, und alle Angaben, die auf mündlichen Auskünften beruhen, sind als solche kenntlich gemacht. Bei den von mir durchgeführten und in der Dissertation erwähnten Untersuchungen habe ich die Grundsätze guter wissenschaftlicher Praxis, wie sie in der „Satzung der Justus-Liebig- Universität Gießen zur Sicherung guter wissenschaftlicher Praxis“ niedergelegt sind, eingehalten. Gießen, den (Ines Fonfara) 3 Danksagung DANKSAGUNG Zuerst möchte ich mich bei Prof. Dr. Alfred Pingoud für die Möglichkeit in seinem Institut Doktorarbeit machen zu dürfen bedanken. Danke auch für die wertvollen Diskussionen, Ideen und die stetige Motivation. Herrn Prof. Dr. Michael Martin danke ich für die Übernahme des Koreferats. Dr. Wolfgang „Wolle“ Wende danke ich für die langjährige Betreuung, das stetig offene Ohr, die Kritik, die einen anspornt doch immer nochmal ein bisschen besser zu sein, und die grenzenlose Geduld. Prof. Dr. Peter Friedhoff gilt mein Dank für die zahlreichen Diskussionen, teilweise zwischen „Tür und Angel“, für die vielen kleinen und großen Hilfestellungen und für die Möglichkeit Mitglied des IRTG gewesen zu sein. Meinen russischen Kollaborationspartnern Dr. Andrei Alexeevski und Alexander Grishin danke ich für die Möglichkeit bei Ihnen etwas über Bioinformatik zu lernen. Danke an Dr. Ute Curth, für die Hilfe bei den AUZ Experimenten. Ein besonderer Dank an Karina und Ina, die bei allen bürokratischen Schwierigkeiten stets zur Seite standen und alles geregelt haben und ein ganz großes Dankeschön an Anja, für die tolle Organisation rund um das IRTG und natürlich für das Korrekturlesen. Ich möchte mich ausdrücklich beim gesamten Institut für Biochemie für die ausgesprochen nette Arbeitsatmosphäre bedanken. Vor allem bei den „alten“ Mitgliedern, die mich in Ihren Reihen aufgenommen und immer unterstützt haben und die zu guten Freunden geworden sind. Steffi: danke für die vielen Gespräche und Unternehmungen innerhalb und außerhalb des Labors, Katja: danke für die kleinen und großen „Pausen“, Ann-Jo: danke für den vielen Spaß im Institut und natürlich auch danach und die immer wieder interessanten Diskussionen, Roger: danke für die verrückte Zeit in Moskau und die seelisch und moralische Unterstützung und Jasmina danke für die schöne Zeit zusammen im L1 und vor allem auch danach. Danke, dass ich euch kennenlernen durfte. 4 Danksagung Außerdem danke ich Ina D., die mich in die Geheimnisse der Laborarbeit eingeweiht hat, Sabrina für die technische Unterstützung und den Spaß zwischendurch und Caro für die super Zeit in „unserem“ Labor, ich hätte mir keine bessere Laborkollegin wünschen können. Ein besonderes Dankeschön geht an Dr. Vera Pingoud die mir viele kleine Tricks und Kniffe beigebracht hat. Meinen Freunden Jessica, Fenja, Judith, Janette, Stöps, Susi, Falk und David möchte ich dafür danken, dass sie immer an mich geglaubt haben, immer für mich da waren und ein offenes Ohr hatten und mir immer wieder gezeigt haben das es auch noch was anderes gibt als Arbeit. Dankeschön, ich weiß nicht, was ich ohne euch gemacht hätte. Abschließend gilt mein Dank meinen Eltern und meinem Bruder für die Unterstützung, die Liebe und das in mich gesetzte Vertrauen, dass sie mir immer den Rücken gestärkt haben und mir damit das alles erst ermöglich haben. Dankeschön für euer Verständnis, als ich mich für diesen Weg entschieden habe. 5 “O verdadeiro encenador da nossa vida é o acaso – um encenador cheio de crueldade, misericórdia e encanto cativante.” Amadeu de Prado – Um ourives das palavras „Der wirkliche Regisseur unseres Lebens ist der Zufall - ein Regisseur voll der Grausamkeit, der Barmherzigkeit und des bestrickenden Charmes.“ Amadeu de Prado – Der Goldschmied der Worte (Pascal Mercier – Nachtzug nach Lissabon) 6 PUBLICATIONS Grishin A., Fonfara I., Alexeevski A., Spirin S., Zanegina O., Karyagina A., Alexeyevsky D., Wende W. Identification of conserved features of LAGLIDADG homing endonucleases. J Bioinform Comput Biol, 2010 Jun; 8 (3):453-69 Fonfara I., Curth U., Pingoud A., Wende W., Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases, Nucleic Acids Res, 2011; doi: 10.1093/nar/gkr788 PATENT APPLICATION Application No 61/495410 “Nuclease fusion protein and uses thereof” ORAL PRESENTATIONS August 2008, SPP1170 Workshop, Bremen, Germany, “New strategies to generate homing endonucleases of pre-defined specificity by directed evolution” November 2009, Minisymposium IRTG 1384, Moscow, Russia, “A new approach for generating meganucleases” May 2010, Workshop IRTG 1384 “Enzymes and enzyme complexes acting on nucleic acids”, Vilnius, Lithuania, “Natural and artificial meganucleases – tools for gene therapy” June 2010, Off-Spring Meeting IRTG 1384, Moscow, Russia, “Binding and bending of DNA by proteins examined via gel-based assays” POSTER PRESENTATIONS June 2010, FASEB Summer Research Conference “Nucleic Acid Enzymes”, Saxton River, Vermont, USA, “Extending meganucleases via domain fusions” August 2010, 6th NEB Meeting on DNA Restriction and Modification, Bremen, Germany, “Natural and artificial meganucleases – tools for gene therapy” 7 Table of contents TABLE OF CONTENTS ABBREVIATIONS ................................................................................................................ 11 1. INTRODUCTION .......................................................................................................... 12 1.1 GENE TARGETING ......................................................................................................... 12 1.1.1 Homing endonucleases ......................................................................................... 13 1.1.2 Zinc-finger nucleases ........................................................................................... 17 1.1.3 TALE nucleases .................................................................................................... 19 1.1.4 TFO-linked nucleases ........................................................................................... 20 1.2 MEGANUCLEASES .......................................................................................................... 20 1.2.1 Directed Evolution ............................................................................................... 20 1.2.1.1 PI-SceI .......................................................................................................... 22 1.2.2 Fusion enzyme engineering .................................................................................. 24 1.2.2.1 I-SceI ............................................................................................................ 24 1.3 AIM ............................................................................................................................... 24 2. MATERIAL AND METHODS ..................................................................................... 26 2.1 MATERIAL ..................................................................................................................... 26 2.1.1 Chemicals ............................................................................................................. 26 2.1.2 Enzymes ................................................................................................................ 27 2.1.3 Oligonucleotides ................................................................................................... 28 2.1.4 Markers ................................................................................................................ 32 2.1.5 E.coli strains ......................................................................................................... 32 2.1.6 Buffers .................................................................................................................. 33 2.1.6.1 Protein Purification Buffer ........................................................................... 33 2.1.6.2 Cleavage buffer ............................................................................................ 34 2.1.7 Plasmids ............................................................................................................... 35 2.1.7.1 Selection assay
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