Tyrosine site-specific recombinases (Y-SSRs) are DNA editing enzymes that play a valuable role for the manipulation of genomes, due to their precision and versatility. They have been widely used in biotechnology and molecular biology for various applications, and are slowly finding their spot in gene therapy in recent years. However, the limited number of available Y-SSR systems and their often narrow target specificity have hindered the full potential of these enzymes for advanced genome engineering. In this PhD thesis, I conducted a comprehensive investigation of novel Y-SSRs and their potential for advancing genome engineering. This PhD thesis aims to address the current limitations in the genetic toolbox by identifying and characterizing novel Cre-type recombinases and demonstrating their impact on the directed evolution of designer recombinases for precise genome surgery. To achieve these aims, I developed in a collaboration a comprehensive prediction pipeline, combining a rational bioinformatical approach with knowledge of the biological functions of recombinases, to enable high success rate and high-throughput identification of novel tyrosine site-specific recombinase (Y-SSR) systems. Eight putative candidates were molecularly characterized in-depth to ensure their successful integration into future genome engineering applications. I assessed their activity in prokaryotes (E. coli) and eukaryotes (human cell lines), and determined their specificity in the sequence space of all known Cre- type target sites. The potential cytotoxicity associated with cryptic genomic recombination sites was also explored in the context of recombinase applicability. This approach allowed the identification of novel Y-SSRs with distinct target sites, enabling simultaneous use of multiple Y-SSR systems, and provided knowledge that will facilitate the assignment of novel and known recombinases to specific uses or organisms, ensuring their safe and effective implementation. The introduction of these novel Y-SSRs into the genome engineering toolbox opens up new possibilities for precise genome manipulation in various applications. The broader targetability offered by these enzymes could accelerate the development of novel gene therapies, as well as advance the understanding of gene function and regulation. Moreover, these recombinases could be used to design custom genetic circuits for synthetic biology, allowing researchers to create more complex and sophisticated cellular systems. Finally, I introduced the novel Y-SSRs into efforts aimed at developing designer recombinases for precise genome surgery, demonstrating their impact on accelerating the directed evolution process. Therapeutically relevant recombinases with altered DNA specificity have been developed for excision or inversion of specific DNA sequences. However, the potential for evolving recombinases capable of integrating large DNA cargos into naturally occurring lox-like sites in the human genome remained untapped so far. Thus, I embarked on evolving the Vika recombinase to mediate the integration of DNA cargo into a native human sequence. I discovered that Vika could integrate DNA into the voxH9 site in the human genome, and then, I enhanced the process through directed evolution. The evolved variants of Vika displayed a marked improvement in integration efficiency in bacterial systems. However, the translation of these results into mammalian systems has not yet been entirely successful. Despite this, the study laid the groundwork for future research to optimize the efficiency and applicability of Y-SSRs for genomic integration. In summary, this thesis made significant strides in the identification, characterization, and development of novel Y-SSRs for advanced genome engineering. The comprehensive prediction pipeline, combined with in-depth molecular characterization, has expanded the genetic toolbox to meet the growing demand for better genome editing tools. By exploring efficiency, cross-specificity, and potential cytotoxicity, this research lays the foundation for the safe and effective application of novel Y-SSRs in various therapeutic settings. Furthermore, by demonstrating the potential of these recombinases to improve efforts in creating designer recombinases through directed evolution, this research has opened new avenues for precise genome surgery. The successful development and implementation of these novel recombinases have the potential to revolutionize gene therapy, synthetic biology, and our understanding of gene function and regulation.

Die Modifikation von Genen ist in den molekularen Biowissenschaften ein fundamentales Werkzeug, um die Funktion von Genen zu studieren (Reverse Genetik). Diese Arbeit hat erfolgreich Zinkfinger- und CRISPR/Cas9-Nukleasen für die Verwendung in C. reinhardtii etabliert, um Gene im Kerngenom gezielt auszuschalten und präzise zu verändern. Basierend auf vorausgegangener Arbeit mit Zinkfingernukleasen (ZFN) konnte die Transformationseffizienz um das 300-fache verbessert werden, was die Inaktivierung von Genen auch in motilen Wildtyp-Zellen ermöglichte. Damit war es möglich, die Gene für das Kanalrhodopsin-1 (ChR1), Kanalrhodopsin-2 (ChR2) und das Chlamyopsin-1/2-Gen (COP1/2) einzeln und gemeinsam auszuschalten. Eine Analyse der Phototaxis in diesen Stämmen ergab, dass die Phototaxis durch Inaktivierung von ChR1 stärker beeinträchtigt ist als durch Inaktivierung von ChR2. Um das CRISPR/Cas9-System zu verwenden, wurden die Transformationsbedingungen so angepasst und optimiert, dass der Cas9-gRNA-Komplex als in vitro hergestelltes Ribonukleoprotein in die Zellen transformiert wurde. Um die Bedingungen für präzise Genmodifikationen zu messen und zu verbessern, wurde das SNRK2.2-Gen als Reportergen für eine „Blau-Grün Test“ etabliert. Kleine Insertionen von bis zu 30 bp konnten mit kurzen Oligonukleotiden eingefügt werden, während größere Reportergene (mVenus, SNAP-Tag) mithilfe eines Donor-Plasmids generiert wurden. In dieser Arbeit konnten mehr als 20 nicht-selektierbare Gene – darunter 10 der 15 potenziellen Photorezeptorgene – mit einer durchschnittlichen Mutationsrate von 12,1 % inaktiviert werden. Insgesamt zeigt diese Arbeit in umfassender Weise, wie Gen-Inaktivierungen und Modifikationen mithilfe von ZFNs und des CRISPR/Cas9-Systems in der Grünalge C. reinhardtii durchgeführt werden können. Außerdem bietet die Sammlung der zehn Photorezeptor-Knockouts eine aussichtsreiche Grundlage, um die Vielfalt der Photorezeptoren in C. reinhardtii zu erforschen. / Gene editing is a fundamental tool in molecular biosciences in order to study the function of genes (reverse genetics). This study established zinc-finger and CRISPR/Cas9 nucleases for gene editing to target and inactivate the photoreceptor genes in C. reinhardtii. In continuation of previous work with designer zinc-finger nucleases (ZFN), the transformation efficiency could be improved 300-fold, which enabled the inactivation of genes in motile wild type cells. This made it possible to disrupt the Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2) and Chlamyopsin-1/2 (COP1/2) genes individually and in parallel. Phototaxis experiments in these strains revealed that the inactivation of ChR1 had a greater effect on phototaxis than the inactivation of ChR2. To apply the CRISPR/Cas9 system, the transformation conditions were adapted and optimized so that the Cas9-gRNA complex was successfully electroporated into the cells as an in vitro synthesized ribonucleoprotein. This approach enabled gene inactivations with CRISPR/Cas9 in C. reinhardtii. In order to measure and improve the conditions for precise gene modifications, the SNRK2.2 gene was established as a reporter gene for a ‘Blue-Green test’. Small insertions of up to 30 bp were inserted using short oligonucleotides, while larger reporter genes (mVenus, SNAP-tag) were integrated using donor plasmids. Throughout this study, more than 20 non-selectable genes were disrupted, including 10 of the photoreceptor genes, with an average mutation rate of 12,1 %. Overall, this work shows in a comprehensive way how gene inactivations and modifications can be performed in green alga C. reinhardtii using ZFNs or CRISPR/Cas9. In addition, the collection of the ten photoreceptor knockouts provides a promising source to investigate the diversity of photoreceptor genes in C. reinhardtii.