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Engineering the Genome of Minimal Bacteria Using CRISPR/Cas9 Tools Iason Tsarmpopoulos

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Engineering the Genome of Minimal Bacteria Using CRISPR/Cas9 Tools Iason Tsarmpopoulos Engineering the genome of minimal bacteria using CRISPR/Cas9 tools Iason Tsarmpopoulos To cite this version: Iason Tsarmpopoulos. Engineering the genome of minimal bacteria using CRISPR/Cas9 tools. Mi- crobiology and Parasitology. Université de Bordeaux, 2017. English. NNT : 2017BORD0787. tel- 01834971 HAL Id: tel-01834971 https://tel.archives-ouvertes.fr/tel-01834971 Submitted on 11 Jul 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE PRÉSENTÉE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE BORDEAUX ÉCOLE DOCTORALE Science de la vie et de la Santé SPÉCIALITÉ Microbiologie and Immunologie Par Iason TSARMPOPOULOS Ingénierie de génome de bactéries minimales par des outils CRISPR/Cas9 Sous la direction de : Monsieur Pascal SIRAND-PUGNET Soutenue le jeudi 07 décembre 2017 à 14h00 Lieu : INRA, 71 avenue Edouard Bourlaux 33882 Villenave d'Ornon salle Amphithéâtre Josy et Colette Bové Membres du jury : Mme Cécile BEBEAR Université de Bordeaux et CHU de Bordeaux Président Mme Florence TARDY Anses-Laboratoire de Lyon Rapporteur M. Matthieu JULES Institut Micalis, INRA and AgroParisTech Rapporteur M. David BIKARD Institut Pasteur Examinateur M. Fabien DARFEUILLE INSERM U1212 - CNRS UMR 5320 Invité Mme Carole LARTIGUE-PRAT INRA - Université de Bordeaux Invité M. Pascal SIRAND-PUGNET Université de Bordeaux Directeur de thèse Ingénierie de génome de bactéries minimales par des outils CRISPR/Cas9 Les mycoplasmes sont des bactéries pathogènes, dotées de petits génomes d’environ 1Mbp, avec une faible teneur en G+C. L'intérêt de la communauté scientifique pour ces bactéries a été récemment renouvelé par des avancées dans les domaines de la synthèse et de la transplantation de génomes. Ces nouvelles approches ont ouvert la voie à l'ingénierie génomique à grande échelle des mycoplasmes. Les systèmes CRISPR/Cas sont des systèmes de défense adaptatifs procaryotes contre les acides nucléiques invasifs. Le système CRISPR de Streptococcus pyogenes est composé d’une endonucléase (SpCas9) et de deux CRISPR ARNs (crRNA et tracrRNA) qui dirigent Cas9 vers sa séquence d’ADN cible. La reconnaissance de l’ADN cible se fait par appariement du crRNA et de la présence en aval d’une séquence nommée protospacer adjacent motif (PAM). Apres cette reconnaissance, Cas9 coupe l’ADN cible. A partir de ce système, un outil génétique simplifié composé de Cas9 et d’un ARN guide (gRNA) a été développé pour de nombreux organismes. Le premier objectif de ma thèse était de combiner les méthodes de biologie synthétique de clonage et de la transplantation de génomes avec les outils CRISPR/Cas9 pour l’ingénierie des génomes de mycoplasmes clonés dans la levure. Nous avons réussi à utiliser cette approche pour enlever des gènes et des régions génomiques dans trois espèces: Mycoplasma mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum et M. pneumoniae. Afin de développer un système plus adapté aux mycoplasmes, nous avons ensuite caractérisé le système CRISPR/Cas9 de Mycoplasma gallisepticum (Mg). En utilisant une combinaison d'approches in silico et in vivo, la séquence PAM de MgCas9 a été caractérisée comme NNNAAAA. Nous avons alors entrepris de développer un système CRISPR/Cas minimal de M. gallisepticum pour une utilisation directe dans les cellules de mollicutes: le gène codant MgCas9 a été introduit dans le génome de Mmc, mais son activation avec un gRNA chimère entre le crRNA et le tracrRNA de M. gallisepticum n’a pas été obtenue pour le moment. Mots clés : Biologie de synthèse, CRISPR/Cas9, Mycoplasma Engineering the genome of minimal bacteria using CRISPR/Cas9 tools Mycoplasmas are small pathogenic bacteria that are characterized by reduced genomes of about 1 Mbp with a low G+C content. The interest of the scientific community towards these species has been recently renewed by successful synthesis of their genome and transplantation experiments. These new genetic tools opened the way to further applications and developments for large-scale genome engineering programmes. CRISPR/Cas systems are natural systems that provide bacteria and archaea with an adaptive defense mechanism against invading nucleic acids. The CRISPR system from Streptococcus pyogenes includes an endonuclease (SpCas9) and two CRISPR RNAs (crRNA et tracrRNA) which role are to drive Cas9 to a target sequence. Target recognition depends on a specific pairing of the crRNA and the presence of a motif named protospacer adjacent motif (PAM). After recognition, Cas9 cleaves the targeted DNA. From the natural S. pyogenes system, a simplified genetic tool including Cas9 and a guide RNA (gRNA) was developed for many organisms . The first goal of my thesis was to combine the synthetic biology methods of genome cloning in yeast and back transplantation into recipient cells with a CRISPR/Cas9 tool for efficient engineering of mycoplasma genomes cloned in yeast. We succeeded in removing genes and genomic regions in three different species, Mycoplasma mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum and M. pneumoniae. Then, in order to develop a system optimized for mycoplasma genome editing, we characterized a natural CRISPR/Cas9 system derived from Mycoplasma gallisepticum (Mg). Using a combination of in silico and in vivo approaches, MgCas9 PAM sequence was characterized as NNNAAAA. We then started to develop a minimal CRISPR/Cas system from M. gallisepticum for direct genome editing in mollicutes. Thus we introduced MgCas9 encoding gene in Mmc and tried to activate it with a newly designed gRNA, a chimeric molecule between the crRNA and the tracrRNA of M. gallisepticum, without success yet. Keywords : Synthetic Biology, CRISPR/Cas9, Mycoplasma UMR 1332 Biologie du Fruit et Pathologie INRA, IBVM, 71 Av. E. Bourlaux, CS 20032 33882 Villenave d'Ornon Cedex, France Table of contents Acronym List p.1 Introduction p.2 I. Mollicutes p.2 II. Taxonomy and Phylogeny p.3 a. Taxonomy p.3 b. Phylogeny p.4 III. Evolution and structure of mollicutes genome p.4 i. Evolution with genome reduction p.4 ii. Synteny loss p.5 iii. Impact of horizontal gene transfer p.5 iv. HGT in mollicutes p.6 1. Between human pathogens p.6 2. Between bird pathogens p.6 3. Between ruminant pathogen p.7 IV. Mollicutes mobile elements p.8 a. Insertion sequences p.8 b. Integrative and conjugative elements p.8 c. Plasmids in Mollicutes p.10 d. Phages p.10 e. Mobile element control p.11 Restriction-modification systems p.11 Abortive infection mechanisms and Bacteriophages Exclusion system p.12 CRISPR/Cas systems p.12 V. Pathogenicity and disease control p.12 VI. Genome engineering of mollicutes p.16 a. Random mutagenesis using transposons p.16 b. Directed mutagenesis p.17 i. Directed mutagenesis using suicide plasmids p.18 ii. Directed mutagenesis using oriC plasmids p.18 c. Synthetic biology applications p.19 i. Cloning natural or synthetic genomes of mycoplasma in yeast p.19 ii. Genome transplantation as a key process to get a living bacterium p.20 iii. Tools for genome engineering of bacterial genome cloned in yeast p.21 VII. CRISPR/Cas system p.22 a. Natural prokaryotic system p.22 b. Functional characterization of the CRISPR system p.23 c. CRISPR as a gene regulation mechanism p.24 d. Universal tool for genome manipulation p.24 e. Development of CRISPR/Cas system as an editing tool for bacterial p.26 Objectives of thesis p.27 Results Chapter 1: Adaptation of the CRISPR/Cas9 of Streptococcus pyogenes for manipulation of mycoplasma genome already transformed in yeast p.28 A. Strategy p.28 B. Application of CRISPR/Cas9 for genome editing in Saccharomyces cerevisiae p.29 C. Proof of concept: Replacement of glpO gene with a marker in Mmc p.29 D. Evaluation of the CRISPR/Cas9 tool for in-yeast engineering of different mycoplasma species p.32 a. M. capricolum subsp. capricolum: precise deletions within the MCAP0015- MCS2-MCAP0017 locus p.32 b. M. pneumoniae: deletion of a virulence factor (MPN142) p.33 Discussion p.34 Chapter 2: Characterization of the native CRISPR/Cas9 system of M. gallisepticum p.36 Comparative genomics of CRISPR in mollicutes p.36 1. Distribution of CRISPR system in mollicutes p.36 2. Phylogenomics of mollicutes CRISPR systems p.37 3. Direct Repeats and tracrRNA p.38 4. CRISPR of M. gallisepticum p.39 5. In silico prediction of PAM sequence p.40 o Development of a dedicated “R” script p.40 o Particular case of spacer 12 of M. gallisepticum R low and R high strains p.41 o Extra Candidates collection p.41 6. In vivo evaluation of PAM candidates and CRISPR/Cas9 system activity in M. gallisepticum p.42 o One and two plasmids strategies p.42 o Determination of PAM consensus sequence p.44 Discussion p.45 Chapter 3. M. gallisepticum CRISPR system as a tool for targeted cleavage in mollicutes p.47 o Introduction of a MgCas9 gene in the Mmc genome p.47 o Construction of a hybrid gRNA from Mgal CRISPR p.48 o Evaluation of the MgCas9/gRNA tool in Mmc p.49 o Transformation of the MmcCas9ICE cells with the gRNA p.49 Discussion p.49 General Discussion p.51 Chapter 1 p.51 Chapter 2 p.53 Chapter 3 p.56 Materials and Methods p.59 Abbreviation list A, C, G, T, U = Adenine, Cytosine, Guanine, Thymine, Uracil Cas proteins = CRISPR associated proteins CDS = Coding DNA Sequence CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats crRNA = Crispr RNA DNA = Deoxyribo-Nucleic Acid DR = Direct Repeat DSB(s) = Double strand Break(s) glpO = glycerol-3-phosphate oxidase gRNA = guide RNA HCO = Hexaammine-CObalt HDR = Homologous Directed Repair HGT = Horizontal Gene Transfer ICE = Integrative and Conjugative Element Mcap = Mycoplasma capricolum subsp.
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