Master of Science / Department of Biomedical Sciences / Raymond R. R. Rowland / Porcine Reproductive and Respiratory Syndrome Virus (PRSSV) is one of the most economically important diseases in the global swine industry, costing producers an estimated $660 million annually. PRRSV is genetically diverse with a low replication fidelity, due to it being an RNA virus, resulting in multitudes of isolates being produced. This virus has a tropism for cells of the monocyte/macrophage lineage. Cluster of Differentiation 163 (CD163) is considered the primary PRRSV receptor located on porcine alveolar macrophages (PAMs). CRISPR/Cas9 technology was utilized to knock out CD163 via a frameshift mutation, resulting in pigs of the CD163 Null genotype. Also, a domain of porcine CD163 was deleted and replaced with the insertion of a CD163 homolog of human-like domain and neomycin cassette to serve as a genetic marker. This swap resulted in pigs that possessed a CD163L1 domain 8 mimic of porcine homolog human CD163-like (hCD163L-1) of SRCR domain 8. Previous work has demonstrated that CD163 Null pigs were resistant to one genotype 2 PRRSV isolate. An in vivo study was performed to observe whether hCD163L-1 pigs were also resistant to infection. Various diagnostic tests were performed to determine the presence or absence of PRRSV viremia levels in serum, CD163 receptor surface expression levels on PAMs, IgG antibody levels and haptoglobin (Hp) levels in serum. hCD163L-1 pigs did not support genotype 1 PRRSV replication, but were susceptible to genotype 2 PRRSV infections. In addition, in vitro infection experiments were performed on PAMs and macrophages derived from peripheral blood mononuclear cells (PBMCs) to determine resistance to multiple isolates. hCD163L-1 macrophages showed reduced infection with genotype 2 and no infection with genotype 1 PRRSV during in vitro infections. Null PAMs and PBMCs derived macrophages did not support infection towards any isolate of either PRRSV genotype.

Yes / Integrating a desired DNA sequence into the yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans. / This work was supported by the Wellcome Trust [202062 to B.H. and 207127 to A.M.] This work was also supported by a SURE studentship from the University of Sheffield [325537] awarded to P.W.D

O fator de transcrição A mitocondrial (TFAM) é um membro da subfamília HMGB que se liga a promotores do DNA mitocondrial (mtDNA). É um gene importante para a manutenção do mtDNA, pois regula o número de cópias e é essencial para inicialização da replicação e transcrição do mtDNA. Recentemente técnicas de edição gênica vêm sendo utilizada como uma ferramenta bastante eficaz na manipulação genômica. A nova tecnologia chamada de CRISPR/ Cas9 (Regulary interspaced clustered short palindromic repeats) utiliza um RNA guia (gRNA) curto que contém 20 nucleotídeos complementares a sequência de DNA. Quando o RNA guia se liga ao local alvo, a proteína Cas9 é recrutada para se ligar no local alvo e induzir a dupla quebra na cadeia de DNA. Neste contexto, este estudo propôs editar o gene TFAM pela tecnologia CRISPR Cas9, com o objetivo de gerar células Rho zero através do knock-out em fibroblastos bovinos. Os fibroblastos bovinos utilizados neste estudo foram derivados de uma biopsia de pele coletada de animais adultos. A sequência do gene foi obtida a partir do banco de dados GenBank (www.ncbi.nlm.nih.gov) e esta foi inserida no site CRISPR direct (crispr.dbcls.jp) e no site rgenome (rgenome.net) a fim de desenhar o gRNA. O gRNA foi desenhado no exon 1 do gene TFAM bovino. Os fibroblastos foram cultivados e após as células atingirem 80% de confluência, estas foram eletrotransfectadas com Cas9 (Addgene 48668), gRNA, GFP e plasmídeo controle. Foi utilizado o kit Primary Mammalian Fibroblasts (VPI-1002) e a transfecção foi realizada no equipamento AMAXA Nucleofector 2B. Após a transfecção foi realizada a citometria de fluxo para avaliar a taxa de transfecção, e as células pós transfectadas foram plaqueadas em placas de 96 poços, pela técnica de sorting. O sorting separarou uma célula por poço de 96. Após 20 dias em cultura essas células foram tripsinizadas em placas de 6 poços e o DNA genômico foi extraído, utilizando o kit Qiamp DNA microkit-Qiagen. Para avaliar a frequência de mutações, foi realizada a digestão com a enzima T7 endonuclease, e após confirmado mutações, os clones foram enviados para analise de sequenciamento. Observamos uma taxa de transfecção eficiente de 51,3%. Obtivemos 40 clones com DNA extraído para analise, no qual 7 destes possuiam mutações no local de inserção da CRISPR Cas 9. Com isso, concluimos uma heterozigose mostrando que o desenho da CRISPR foi eficiente, gerando uma deleção do gene TFAM. / The mitochondrial transcription factor A (TFAM) is a member of HMGB subfamily that binds to promoters of mtDNA. It is a very important gene that maintains mtDNA, regulates the number of copies and is essential for the initiation of transcription mtDNA. Recently, gene edition techniques have been used as a very effective tool in genomic manipulation. The new technology called CRISPR/Cas9 (Regulary interspaced clustered short palindromic repeats) uses a short gRNA containing 20 nucleotides complementary to the DNA sequence. When gRNA binds to the target site, the Cas9 protein is recruited to bind in the chosen location and induce double strands breaks in DNA. In this context, this study proposed to edit the TFAM gene by CRISPR Cas9 technology aiming to generate Rho zero cell through the knock-out in bovine fibroblasts. Bovine fibroblasts used in this study were derived from a skin biopsy collected from an adult. The sequence obtained from the database GenBank (www.ncbi.nlm.nih.gov) was inserted in the CRISPR direct site (crispr.dbcls.jp) and in the rgenome site (rgenome.net) to design the RNA guide. The gRNA was designed in the CRISPR direct site (crispr.dbcls.jp) for the Exon 1 of the gene TFAM bovine and after was performed the CRISPR cloning. The fibroblast were cultured and after reaching 80% of confluence, were electro-transfected with Cas9 (Addgene 48668) and control plasmids using the Nucleofector TM Kit for Primary Mammalian Fibroblasts (VPI-1002) and transfected with Cas 9 (Addgene 48668), GFP and control plasmid. Were used the Primary Mammalian Fibroblasts (VPI-1002) and the transfection was performed on the AMAXA Nucleofector 2B. Post transfected cells were analyzed by flow cytometry to evaluate the rate of transfection. The cells post transfected were further split into 1 cell/well (96- well plates for cell cloning). After days in culture these cells were trypsinized in 6-well plates and the genomic DNA was extracted using the Qiamp DNA microkit- Qiagen. To assess the mutation frequency, T7 endonuclease assay were performed and after confirmed the mutations, the clones were sent for sequencing analysis. We observed that the cells were efficiently transfected since they have a rate of 51,3% transfection. We obtained 40 clones with extracted for analysis, in which 7 of these had mutations at the insertion site of CRISPR/Cas 9. We concluded that until this moment the CRISPR design was efficient and that we obtained a deletion of the TFAM gene.

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. / 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.

O fator de transcrição A mitocondrial (TFAM) é um membro da subfamília HMGB que se liga a promotores do DNA mitocondrial (mtDNA). É um gene importante para a manutenção do mtDNA, pois regula o número de cópias e é essencial para inicialização da replicação e transcrição do mtDNA. Recentemente técnicas de edição gênica vêm sendo utilizada como uma ferramenta bastante eficaz na manipulação genômica. A nova tecnologia chamada de CRISPR/ Cas9 (Regulary interspaced clustered short palindromic repeats) utiliza um RNA guia (gRNA) curto que contém 20 nucleotídeos complementares a sequência de DNA. Quando o RNA guia se liga ao local alvo, a proteína Cas9 é recrutada para se ligar no local alvo e induzir a dupla quebra na cadeia de DNA. Neste contexto, este estudo propôs editar o gene TFAM pela tecnologia CRISPR Cas9, com o objetivo de gerar células Rho zero através do knock-out em fibroblastos bovinos. Os fibroblastos bovinos utilizados neste estudo foram derivados de uma biopsia de pele coletada de animais adultos. A sequência do gene foi obtida a partir do banco de dados GenBank (www.ncbi.nlm.nih.gov) e esta foi inserida no site CRISPR direct (crispr.dbcls.jp) e no site rgenome (rgenome.net) a fim de desenhar o gRNA. O gRNA foi desenhado no exon 1 do gene TFAM bovino. Os fibroblastos foram cultivados e após as células atingirem 80% de confluência, estas foram eletrotransfectadas com Cas9 (Addgene 48668), gRNA, GFP e plasmídeo controle. Foi utilizado o kit Primary Mammalian Fibroblasts (VPI-1002) e a transfecção foi realizada no equipamento AMAXA Nucleofector 2B. Após a transfecção foi realizada a citometria de fluxo para avaliar a taxa de transfecção, e as células pós transfectadas foram plaqueadas em placas de 96 poços, pela técnica de sorting. O sorting separarou uma célula por poço de 96. Após 20 dias em cultura essas células foram tripsinizadas em placas de 6 poços e o DNA genômico foi extraído, utilizando o kit Qiamp DNA microkit-Qiagen. Para avaliar a frequência de mutações, foi realizada a digestão com a enzima T7 endonuclease, e após confirmado mutações, os clones foram enviados para analise de sequenciamento. Observamos uma taxa de transfecção eficiente de 51,3%. Obtivemos 40 clones com DNA extraído para analise, no qual 7 destes possuiam mutações no local de inserção da CRISPR Cas 9. Com isso, concluimos uma heterozigose mostrando que o desenho da CRISPR foi eficiente, gerando uma deleção do gene TFAM. / The mitochondrial transcription factor A (TFAM) is a member of HMGB subfamily that binds to promoters of mtDNA. It is a very important gene that maintains mtDNA, regulates the number of copies and is essential for the initiation of transcription mtDNA. Recently, gene edition techniques have been used as a very effective tool in genomic manipulation. The new technology called CRISPR/Cas9 (Regulary interspaced clustered short palindromic repeats) uses a short gRNA containing 20 nucleotides complementary to the DNA sequence. When gRNA binds to the target site, the Cas9 protein is recruited to bind in the chosen location and induce double strands breaks in DNA. In this context, this study proposed to edit the TFAM gene by CRISPR Cas9 technology aiming to generate Rho zero cell through the knock-out in bovine fibroblasts. Bovine fibroblasts used in this study were derived from a skin biopsy collected from an adult. The sequence obtained from the database GenBank (www.ncbi.nlm.nih.gov) was inserted in the CRISPR direct site (crispr.dbcls.jp) and in the rgenome site (rgenome.net) to design the RNA guide. The gRNA was designed in the CRISPR direct site (crispr.dbcls.jp) for the Exon 1 of the gene TFAM bovine and after was performed the CRISPR cloning. The fibroblast were cultured and after reaching 80% of confluence, were electro-transfected with Cas9 (Addgene 48668) and control plasmids using the Nucleofector TM Kit for Primary Mammalian Fibroblasts (VPI-1002) and transfected with Cas 9 (Addgene 48668), GFP and control plasmid. Were used the Primary Mammalian Fibroblasts (VPI-1002) and the transfection was performed on the AMAXA Nucleofector 2B. Post transfected cells were analyzed by flow cytometry to evaluate the rate of transfection. The cells post transfected were further split into 1 cell/well (96- well plates for cell cloning). After days in culture these cells were trypsinized in 6-well plates and the genomic DNA was extracted using the Qiamp DNA microkit- Qiagen. To assess the mutation frequency, T7 endonuclease assay were performed and after confirmed the mutations, the clones were sent for sequencing analysis. We observed that the cells were efficiently transfected since they have a rate of 51,3% transfection. We obtained 40 clones with extracted for analysis, in which 7 of these had mutations at the insertion site of CRISPR/Cas 9. We concluded that until this moment the CRISPR design was efficient and that we obtained a deletion of the TFAM gene.

Le cancer est responsable de plus de 15% des décès. L’activation d’oncogènes et l’inactivation de gènes suppresseurs de tumeur contribuent à la transformation des cellules. Dans 50% des cas de cancers le gène TP53 est muté. C’est pourquoi comprendre les conséquences de ces mutations est indispensable pour développer des tests permettant de cibler p53 dans le cadre de thérapies. Dans cette étude nous avons utilisé la nouvelle technique de modification de génomes, CRISPR-Cas9. Cette technique a été utilisée dans le but d’introduire des mutations spécifiques de TP53 dans le génome de fibroblastes non tumoraux. Nous avons alors analysé les effets de ces mutations au niveau transcriptionnel et protéomique. Ces analyses aideront à identifier les effets spécifiques de chaque mutation de p53. Ces résultats seront utilisés pour établir des lignées cellulaires permettant de cribler et d’identifier des composés capables de restaurer la fonction sauvage de p53. / Cancer is responsible for more than 15% of human deaths. Activation of oncogenes and inactivation of tumor suppressor genes contribute to malignant transformation of cells. Mutations of the tumor suppressor gene TP53 are observed in about 50% of human cancers. Therefore, it is of high interest to understand functional consequences of TP53 mutations in order to develop biological tests that allow targeting mutant p53 for oncotherapy. In this study we use CRISPR-Cas9, the latest genome editing technique, for introducing specific TP53 mutations into the genome of a non-tumoral fibroblast cell line. We analyze the effects of p53 mutations at the transcriptomic and proteomic level. These analyses will help identifying gene- and pathway-specific effects of distinct p53 mutations. These results will be used for establishing cell lines that allow high throughput screening, in order to discover new chemical compounds that are able to restore crucial functions of mutant p53 proteins.

The first part of the thesis is devoted to the study of an epigenetic regulation of genes in unicellular green microalgae. The influence of environment on DNA methylation level, histones modifications, chromatin structure and RNA interferences are described as well as their effect on metabolome. The work focuses more closely on the effect of the demethylation reagent 5-azacytidine and CdCl2 on the amount of 5-mC and metabolites of the methionine cycle in Chlamydomonas reinhardtii and Scenedesmus quadricauda in the context of the production of secondary metabolites. The second part of the thesis is focused on genetic engineering. Specifically, the CRISPR/Cas9 method was used as RNA-directed gene editing in the model organism, C. reinhardtii. An optimization of the cpFTSY chloroplast gene was performed, demonstrating phenotypic change in mutated colonies. Furthermore, CRISPR/Cas9 was used to edit the adiposuppressor gene WDTC1. For both genes we obtained mutants whose DNA was repaired using both NHEJ and HDR. Using the fluorescence NileRed analysis, two ?WDTC1 colonies demonstrated an increased TAG content. Epigenetic mechanisms should be studied more to understand the adaptation of microalgae to stress and the environment, providing valuable information for knowledge of metabolic pathways, transcription factors. This information can be used for controlled gene editing, resulting in increased amounts of product for the same or better biomass growth.

CRISPR/Cas9 is the gene editing tool of choice in basic research and poised to become one in clinical context. However, current studies on the topic suffer from a number of shortcomings. Mutagenesis is often assessed using bulk methods, which means rare events go undetected, unresolved or are discarded as potential sequencing errors. Many of the genotyping methods rely on short-range PCR, which excludes larger structural variants. Other methods, such as FISH, do not provide basepair resolution, making the genotype assessment imprecise. Furthermore, it is not well understood how Cas9 delivery format influences the dynamics of indel introduction. Finally, many studies of on-target activity were conducted in cancerous cell lines, which do not accurately model the mutagenesis of normal cells in the therapeutic context. In my thesis, I have investigated on-target lesions induced by Cas9 complexed with single gRNAs and no exogenous template. I have followed the time dynamics of Cas9-induced small indels as a function of reagent delivery methods, established an assay for quantification of Cas9-induced genomic lesions that are not small indels ("complex lesions") and used this assay to isolate and genotype complex lesions, many of which would be missed by standard methods. I found that DNA breaks introduced by single guide RNAs frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and cross-over events were identified. Frequent and extensive DNA damage in mitotically active cells caused by CRISPR/Cas9 editing may have pathogenic consequences.

In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.

Eukaryotes use proteins to carefully package and compact their genomes to fit into the nuclei of their individual cells. Nucleosomes are the primary level of compaction. Nucleosomes are formed when DNA wraps around an octamer of histone proteins and a nucleosome's position can limit access to genetic regulatory elements. Therefore, nucleosomes represent a basic level of gene regulation. DNA and its associated proteins, called chromatin, is usually classified as euchromatin or heterochromatin. Euchromatin is transcriptionally active with loosely packed nucleosomes while heterochromatin is condensed with tightly packed nucleosomes and is transcriptionally silent. In order to become active, heterochromatin must first be remodeled. We have studied the effects of nucleosome positioning on transgene expression in vivo using Caenorhabditis elegans as a model. We show that both location and polarity of the DNA sequence can influence transgene expression. We also discuss some considerations for working with CRISPR/Cas9. A major reason for doing in vitro nucleosome reconstitutions is to determine the effects of DNA sequence on nucleosome formation and position. It has previously been implied that nucleosome reconstitutions are stochastic and not very reproducible. We show that nucleosome reconstitutions are highly reproducible under our reaction conditions. Our results also indicate that a minimum depth of 35X sequencing coverage be maintained for maximal gains in Pearson's correlation coefficients. Communicating science with others is an important skill for any researcher. The rising generation of scientists need mentors who can teach them how to be independent thinkers who can carry out scientific experiments and communicate their finding to others. With this goal in mind, we have devised a scaffolding pedagogical method to help transform undergraduates into confident independent thinkers and researchers.