Les réarrangements permettent une évolution rapide du génome par l’acquisition de séquences codantes exogènes, la perte de fonctions non-essentielles ou la création de nouvelles organisations génomiques. Différents mécanismes de réarrangements impliquant des éléments génétiques mobiles (EGM) ont été identifiés chez les archées, les bactéries et les eucaryotes. En revanche, on ignore l’origine des nombreuses inversions génomiques détectées pour les espèces du genre archéen Thermococcus. Mes travaux de thèse visent à améliorer la compréhension de l’évolution génomique chez les Thermococcales à travers l’étude de deux familles d’EGM : les familles de plasmides pTN3 et pT26-2. Plus précisément, je me suis intéressée aux recombinases à tyrosine (ou intégrases) que ces plasmides encodent et qui permettent leur intégration dans le chromosome de l’hôte. J’ai montré que l’intégrase plasmidique Intᵖᵀᴺ³ est responsable d’inversions dans le chromosome de son hôte Thermococcus nautili grâce à une activité catalytique inédite de recombinaison homologue. J’ai par la suite caractérisé deux autres intégrases de Thermococcales reliés phylogénétiquement à Intᵖᵀᴺ³ dont seulement une présente une activité de recombinaison homologue. La comparaison de leurs séquences primaires et la résolution de la structure de Intᵖᵀᴺ³ vont maintenant éclairer les déterminants génétiques responsables de la spécificité de site et de l’activité de recombinaison homologue. Les trois intégrases appartiennent à une classe de recombinases spécifique des archées qui catalyse une intégration suicidaire. Lors de l’intégration, le gène de l’intégrase est fragmenté et probablement désactivé. L’EGM intégré se retrouve piégé dans le chromosome. Les avantages évolutifs d’une telle activité suicidaire restent pour l’instant mystérieux. J’ai identifié 62 intégrases hyperthermophiles suicidaires et reconstruit leur histoire évolutive. Ces intégrases sont très prévalentes et recrutées par différents EGM. De plus, j’ai montré que l’une de ces intégrases présente in vitro une activité de recombinaison site-spécifique à des températures proches de l’ébullition de l’eau, représentant un avantage dans les environnements hyperthermophiles. / Genomes rapidly evolve through rearrangements that can generate new genome organizations or lead to the acquisition of foreign coding sequences or the loss of non-essential functions. Several mechanisms of rearrangement were uncovered for Archaea, Bacteria and Eukaryotes that involve mobile genetic elements (MGE). Species from the archaeal genera Thermococcus present numerous genomic inversions but none of the previously known inversion drivers. To better understand the genomic evolution of Thermococcales, I investigated two of their MGE families: the pTN3 and pT26-2 plasmid families. Specifically, I focused on the tyrosine recombinases (or integrase) that these plasmids encode and that catalyze their site-specific integration in the host chromosome. I demonstrated that the plasmidic integrase Intᵖᵀᴺ³ is responsible for chromosomal inversions in the host Thermococcus nautili through an unprecedented homologous recombination catalytic activity. I also characterized two other related Thermococcus integrases and only one catalyzes homologous recombination. The structure resolution of Intᵖᵀᴺ³ and primary sequence comparisons will now provide clues about the genetic determinants of site specificity and of the homologous recombination activity. The three integrases all belong to an archaeal-specific class of integrases that catalyzes a suicidal integration. The integrase gene is partitioned and presumably inactivated upon integration. The integrated MGE is then trapped into the chromosome. The evolutionary benefits of this suicide activity are puzzling. I identified 62 related suicidal hyperthermophilic integrases and reconstructed their evolutionary history. They are highly prevalent and recruited by diverse MGE. I also showed that one of these integrases can catalyze in vitro site-specific recombination at near boiling water temperature, representing an advantage in hyperthermophilic environments.

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.

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Synthetic biology has ushered in a new age of molecular biology with the aim towards practical developments in disciplines ranging from medicine, agriculture, and industry. Presently, it remains difficult to manipulate the genomes of many organisms due to lack of genetic tools. These problems can be circumvented by cloning large fragments of DNA into strains where many genetic tools are in place, such as Saccharomyces cerevisiae. However, this organism is unable to directly transfer cloned DNA to other organisms and is unable to stably maintain DNA with a G+C content >40%. Many organisms relevant in biotechnology often have G+C content DNA >60%, and therefore are difficult to engineer. Here, the soil bacteria Sinorhizobium meliloti was chosen as a host strain to clone and manipulate large fragments of high G+C content DNA. S. meliloti is a Gram-negativeα-proteobacteria that forms symbiotic relationships with legumes to fix nitrogen. It has a multi-partite genome with a G+C content of 62.7% that includes a chromosome (3.65 Mb), the pSymA (1.35 Mb), and pSymB (1.68 Mb) replicons. A restriction endonuclease hsdR mutant strain lacking pSymA and pSymB was created and used in this study. Multi-host shuttle (MHS) vectors were constructed that allow for direct transfer and maintenance of DNA in E. coli, S. cerevisiae, and P. tricornutum. Characterization of strains was conducted to determine transduction, conjugation, and transformation frequencies, as well as stability of MHS plasmids. Furthermore, a proof-of-concept experiment was conducted to clone large plasmids (70-205 kb) with G+C content >58% via site-specific recombination at a landing pad in the MHS vector, which was then verified using colony PCR. This work demonstrates the usefulness of S. meliloti containing a MHS vector for cloning of large fragments with high G+C content DNA, a technology that may be used for several applications in both applied and basic research. / Thesis / Master of Science (MSc) / Synthetic biology is an emerging field that incorporates principles of molecular biology and engineering for the design and construction of biological systems for application in medicine, agriculture, and industry. Presently, it remains difficult to modify genomes of several organisms due to lack of available techniques. Yeast is currently used for the modification of large DNA pieces, however it is unable to transfer and maintain modified DNA with high G+C content. Here, the bacteria Sinorhizobium meliloti was used as a host organism to conduct genetic engineering due to its ability to maintain large DNA pieces with a high G+C content. Characterization experiments were conducted to assess the efficiency of this organism for this task. Using this strain, a proof-of-concept experiment to demonstrate the uptake and maintenance of large, high G+C DNA pieces was completed. This technology may be useful in biotechnology applications for engineering of large DNA pieces from industrially relevant organisms.

No description available.