Genome editing is an exciting field that allows for the precise modification of an organism's DNA. One of the most advanced tools in this area is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which creates a DSB (Double-strand break) at a specific location in the genome. This break can then be repaired by the cell using one of two pathways – NHEJ (nonhomologous end joining) or HDR (homology-directed repair) HDR leads to more precise repair and is used to create KI (Knock-In) modifications by introducing a homologous piece of DNA with the desired changes. However, HDR is a rare event that competes with the error prone NHEJ pathway, limiting its efficiency. HDR mainly occurs in the G2 and S phases of the cell cycle, making it a challenge to control and target. To improve KI efficiency, researchers have used strategies such as inhibiting NHEJ or activating HDR. This study focuses on identifying direct and indirect activators of HDR through a library assay screening. We established a robust method for screening compounds in HEK293 cells that relies on a plasmid-based delivery Cas9, gRNA (guide RNA), and synthetic ssDNA (single strand DNA). Out of 3,000 compounds screened, 1% showed a higher signal than the positive control, and approximately 10% presented a higher signal than untreated cells. The top 5 compounds were further validated in dose response. Our system opens new avenues for improving the efficiency of KI modifications.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21827号 / 農博第2340号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5199(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 准教授 豊原 治彦, 准教授 田川 正朋 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Although the understanding of genetic predisposition to prostate cancer (PCa) has been improved through genome-wide association studies (GWAS), little is known about the biological implication of germline variants residing in coding or non-coding regions in cancer development and progression. Our hypothesis is that inherited variants may predispose to specific early recurrent genomic events observed in PCa adenocarcinomas, possibly in the context of variable androgen receptor (AR) signaling that changes during a man’s lifetime. Recent in silico analysis by our group on potential association between germline variants and PCa specific somatic lesions identified a non-coding polymorphic regulatory element at the 7p14.3 locus associated with DNA repair and hormone regulated transcript levels and with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08) in human prostate tissue data. In order to functionally characterize the polymorphic 7p14.3 locus (rs1376350, single nucleotide polymorphism, G>A), we set up to establish isogenic cell lines harboring the minor allele by using the CRISPR/Cas9 system. In parallel, CRISPR/Cas9 system was used to knock out different portion of the region encompassing the 7p14.3 variant and to eliminate transcription factors (TFs) binding sites that were identified from previous in silico analysis (i.e. AR and CCAAT/Enhancer Binding Protein (C/EBP) beta (CEBPβ)). The transcriptomes of edited pools and edited single clones from macrodeletion (731 bp), microdeletion (50 bp) and alterations of TFs binding sites were analyzed and compared to the transcriptomes of isogenic cells heterozygous (A/G) and homozygous (A/A) for the minor allele A of the risk variant rs1376350 (with or without AR overexpression). These data identified a set of genes scattered throughout the genome with the same pattern of deregulation suggesting the implication of the variant on the regulation of genes residing in different chromosomes. Additionally, ChIP-qPCR experiments for histone modification supported the identification of the 7p14.3 locus with enhancer activity. Furthermore, ChIP-qPCR of histone mark associated with transcriptional activation or repression in isogenic cells harboring the minor allele A upon AR overexpression showed that the activity of the locus is higher for the minor allele A compared to G, independently from AR activation. Despite the limitations of our model and the current lack of validation in other cells, we confirmed that some of the differentially expressed genes that emerged from the comparative analysis of edited cells are deregulated in human normal and tumor prostate samples as well. This work is a proof of concept of germline predisposition to molecularly distinct cancer subclasses and has the potential to nominate new mechanisms of cancer development. Future work aims to elucidate the mechanisms implicated in the deregulation of the transcriptome by combining the information obtained until now with potential new players that we expect to identify by Mass Spectrometry experiments. To clarify the link between the 7p14.3 variant and the somatic mutations in SPOP, we plan to express mutant SPOP in isogenic cells harboring the minor allele and to asses DNA damage response upon overexpression or silencing of TFs binding at and around the rs1376350 variant. My work is an example of how the CRISPR/Cas9 system can be used to develop a technical framework with convergent approaches to functionally characterize polymorphic regulatory regions including but not limited to the establishment of isogenic cells upon single nucleotide editing.

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25007号 / 医科博第154号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 萩原 正敏, 教授 齋藤 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Mosquitoes belonging to the genera Anopheles transmit malaria parasites, attributing the highest mortality of any vector-borne disease worldwide. Mosquitoes belonging to the genera Aedes transmit arboviruses including dengue, which has become the most important vector-borne virus due to a drastic surge in disease incidence. The scope of the studies in this dissertation is broad, with investigations bringing together elements of classical genetics, recent advances in sequencing and genome-editing technologies, and the use of modern forward genetics approaches. Chapter 2 of this dissertation explores the use of the Oxford Nanopore Sequencing Technology for the first time in mosquitoes. This new technology provides long reads that were used to piece together the AabS3 chromosomal assembly for Anopheles albimanus. The utility of this genomic resource is demonstrated by the discovery of novel telomeric repeats at the ends of the chromosomes that could have important implications in mosquito biology and control. Chapter 3 describes a forward genetics strategy called 'Marker-Assisted Mapping' (MAM) that enables high-resolution mapping of the causal gene locus of a mutant phenotype. The principle and effectiveness of MAM is first demonstrated by mapping a known transgene insertion. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. Genetic crosses between the re mutant and cardinal knocking out individuals generated using CRISPR/Cas9 confirmed that cardinal indeed is the causal gene for re mutation. Chapter 4 explores three innovative strategies for mosquito sex separation by exploiting several sex-linked marker lines. We show that by linking a transgenic marker to the male-determining locus (M locus), or by linking the male-determining Nix gene to a marker, males can be precisely separated from females. We also produce a two-marker transgenic line that allows for both non-transgenic male separation and for efficient line maintenance. Finally, we discuss further applications of the resources generated and future directions stemming from these findings. Altogether, the studies described in this dissertation contribute to the overall goal of understanding mosquito biology and of controlling mosquito-borne infectious diseases. / Doctor of Philosophy / Female mosquitoes bite and transmit deadly pathogens including the malaria parasite, and viruses such as dengue, Zika, and West Nile. Control programs that attempt to limit the spread of these deadly diseases rely on the control of mosquitoes themselves. These mosquito control methods have relied heavily on indoor and outdoor insecticidal spraying. However, the efficacy of these methods has been jeopardized by the increasing prevalence of insecticide resistance. Thus, it is necessary to implement other methods for effective mosquito control. Genetic control strategies such as the Sterile Insect Technique (SIT) and Wolbachia-based Incompatible Insect Technique (IIT) are excellent solutions to overcome the limitations of current control strategies. As female mosquitoes bite and transmit disease-causing pathogens, only males are released, which necessitate the separation of the non-biting males from females before release. The aim of this work was to use recent technological advancements to better understand the genome and basic genetics of vector mosquito species, and to identify possible approaches to improve current sex separation practices. To develop a deep understanding of mosquito biology and genetics, it is crucial that a high-quality and accurate genome assembly is available. However, many mosquito genome assemblies remain fragmented. To address this limitation, we used recent advances in sequencing technologies to produce a high-quality genome assembly for the New World malaria mosquito, Anopheles albimanus. These sequencing and assembly efforts led to the discovery of novel telomere sequences at the ends of chromosomes, which could have implications for mosquito control. Forward genetics, which identifies the gene(s) responsible for a given phenotype, has been hindered by the low recombination rate in the yellow and dengue fever mosquito, Aedes aegypti. We develop a Marker-Assisted Mapping (MAM) strategy to address this problem. We first demonstrate this method by mapping the known insertion of a transgene. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. MAM identification of the Cardinal gene was then verified by knocking out Cardinal, which represents the first successful gene mapping in Aedes aegypti using forward genetics. The MAM strategy has broad implications as it could enable the discovery of genes involved in important traits such as insecticide resistance. To improve sex separation methods, we took advantage of several sex-linked transgenic lines to develop three novel strategies. First, we demonstrate that screening for a genetic marker that is tightly linked to the male-determining locus (M locus) is an effective approach to reduce female contamination. Second, we demonstrate that instead of linking a marker to the M locus, we can link the male-determining factor, Nix, to a genetic marker. When a Nix transgene is located adjacent to the red-eye locus with extremely tight linkage, the red-eye phenotype becomes a faithful marker for separation of males and females. Finally, we developed a two-marker genetic sexing strain that produces non-transgenic males that could be used for release, and transgenic marked males and females for efficient line maintenance.

Fusobacterium nucleatum is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacterium species exhibit genetic recalcitrance, which has led to hindrance in the understanding of their biology and molecular pathogenesis. Though the association of Fusobacterium in diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum ATCC 23726 because it is genetically tractable. Here, in this dissertation, we show that we are able to enhance our existing molecular tools for genome editing to introduce the first mutants in a clinically relevant strain, F. nucleatum ATCC 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cellular invasion and survival. In this work, we identified a novel small adhesin, FadA2, that played a significant role in the invasive ability of F. nucleatum ATCC 25586 to colorectal cancer cells. This dissertation also sheds the first insight into the roles of the type 5a autotransporters. Using a deletion library of genes encoding for the type 5a autotransporter proteins in F. nucleatum ATCC 23726, we systemically characterized altogether 12 type 5a proteins with a focus on the invasion of colorectal cancer cells. Most notably, we found that a wide assortment of type 5a proteins contributing to binding and invasion of F. nucleatum to HCT116 cancer cells. Furthermore, we identified that RadD was not directly involved in inducing secretions of the cytokines IL-8 and CXCL1 while confirmed the specific association of Fap2 in bacterial-induced cytokine secretion. Thus, our findings provided the first comparative and functional analysis of Fusobacterium type 5a autotransporter proteins in colorectal cancer cells which will be crucial to the understanding of Fusobacterium involvement in cancer progression. Finally, this dissertation reported on the first ever observation on the survival strategy of Fusobacterium inside the host cells. We uncovered a novel protein that contributed to enhanced survival of Fusobacterium residing in colorectal cancer cells. This work undoubtedly helps expand the current Fusobacterium genetic toolkit to study proteins and mechanisms relevant to Fusobacterium-accelerated diseases. By identifying and characterizing novel virulence strategies that Fusobacterium can take advantage of, we can increase our comprehension on this opportunistic microbe while devising innovative therapeutic treatments. / Doctor of Philosophy / Fusobacterium, a member of the microbial community in our mouth, has been a captivating study target due to its association with human health and diseases. By nature, Fusobacterium lives in oxygen-free pockets between our teeth and gumline in which this organism has been correlated with a multitude of complications and diseases including periodontitis, inflammatory bowel disease, preterm birth, and most importantly colorectal cancer. Though the connection to human health is established, we still have to learn more about the mechanisms utilized by Fusobacterium to exacerbate diseases. This challenge is mainly hindered by the lack of efficient tools and resources to systematically investigate the relationship between the bacterium and its human host. Therefore, the work in this dissertation focuses on expanding the existing molecular toolkit to study clinically relevant Fusobacterium strain, which provides the power and convenience to discover novel mechanisms that Fusobacterium can take advantage of to be a successful pathogen. Accordingly, we first enhanced our ability to work with a wider range of Fusobacterium species. We successfully introduced exogenous genetic materials into a clinical strain of Fusobacterium, Fusobacterium nucleatum ATCC 25586. This breakthrough was built on the success of our current toolkit to make genetic modifications to a sister strain, Fusobacterium nucleatum ATCC 23726. With this newfound capacity to modify F. nucleatum ATCC 25586, we have described the importance of a novel protein aiding in the invasion of Fusobacterium to colorectal cancer. Furthermore, we have determined that certain proteins within the fusobacterial type 5a protein family can play a key role in governing binding and invasion of colorectal cancer cells in this study. Concurrently, for the first time, we provided the snapshot of a small protein and its role in fusobacterial long-term survival inside its targeted host cells. Altogether, the findings in this dissertation will bring forth an innovative framework to better the comprehension of current Fusobacterium-induced disease implications, while exploring alternative treatments for enhanced patient health.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25316号 / 農博第2582号 / 新制||農||1104(附属図書館) / 京都大学大学院農学研究科農学専攻 / (主査)教授 田尾 龍太郎, 教授 田中 義行, 准教授 中野 龍平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Genome engineering, the deliberate alteration of an organism's genetic material, has revolutionized biotechnology and biomedical research, enabling precise modifications to DNA sequences. Among the tools developed for this purpose, site-specific recombinases (SSRs) stand out for their ability to catalyze targeted DNA rearrangements between defined target sites. The Cre/loxP system, in particular, has been widely used for conditional gene inactivation and recombinase-mediated cassette exchange, facilitating targeted DNA excision, inversion, or integration through the recognition and recombination of loxP target sites. While the inherent specificity of Cre towards the loxP target sequence has been invaluable, it also limits its application to other genomic loci of therapeutic interest. Understanding the factors that govern the enzyme’s DNA specificity opens the possibility to engineer and retarget the complex to non-native sequences, significantly broadening the range of targetable genomic loci. To address this challenge, I describe the development of a high-throughput method to quantify Cre recombination efficiency across a library of loxP-like spacer variants. This method systematically analyzes the impact of spacer sequence alterations to reveal DNA specificity determinants. Through comprehensive screening, the study identified spacer sequences that exhibit inefficient recombination by Cre, despite both full lox sites having matching spacer sequences. Directed evolution was used to enhance Cre activity on these previously 'inert' spacer sequences, generating variants with altered spacer specificity. Detailed molecular analyses, including mutational studies and molecular dynamics simulations, elucidated the structural basis for altered spacer selectivity in evolved Cre variants. The study provides mechanistic insights into the role of specific amino acid residues in determining spacer specificity and highlights the potential for the rational design of recombinases with tailored spacer preferences. Building upon this foundation, I describe the engineering of heterospecific Cre-type SSRs capable of recombining asymmetric DNA target sites. By combining two evolved Cre variants with unique half-site specificities, a functional heterotetrameric complex forms, capable of excising DNA fragments flanked by asymmetric target sequences naturally occurring in the human genome. This approach expands the applicability of SSRs and holds promise for correcting chromosomal inversions underlying genetic disorders, as demonstrated in the correction of the int1h inversion associated with hemophilia A. However, harnessing the full potential of heterospecific SSRs presents challenges, particularly concerning off-target effects resulting from the formation of undesired functional homotetrameric complexes. To mitigate these risks, I investigated strategies to render SSR monomers functionally active in heterotetrameric, but not homotetrameric complexes. Through substrate-linked directed evolution, I identified mutations that confer obligate heterospecificity, leading to safer and more precise genome engineering applications. Together, these studies highlight the transformative potential of engineered SSRs in genome editing and underscore the importance of ongoing research efforts to enhance their specificity, efficacy, and safety for therapeutic interventions and biotechnological applications. By manipulating the highly specific Cre/loxP complex to retarget different lox sequences and analyzing evolved or naturally occurring recombinase recombination specificity, we can better understand how these enzymes can be optimized for therapeutic applications. Furthermore, the ability to confer obligate heterospecificity increases the overall safety of these engineered SSRs, expanding their potential applications in genome engineering, particularly for therapeutic targets that require editing asymmetric (non-palindromic) target sites.

Candida glabrata est une levure pathogène opportuniste, apparaissant aujourd’hui comme la deuxième cause de candidémie en Europe et en Amérique du Nord. Cette levure présente de nombreuses particularités génomiques telles que la présence de nouveaux domaines structuraux au sein d’ARN non-codants ubiquitaires. Le premier aspect de cette thèse a consisté en l’étude de la sous-unité ARN atypique de la Ribonucléase P nucléaire de C. glabrata. Cet ARN contient trois grand domaines additionnels octroyant au transcrit une taille trois fois plus élevée que la moyenne des sous-unités ARN des RNase P eucaryotiques. Les expériences réalisées ont permis une meilleure compréhension du rôle de ces domaines additionnels et ont démontré la présence inédite de la protéine Rcl1 au sein du complexe de la RNase P. Dans un second temps ce travail de thèse a aussi contribué à l’amélioration des outils d’édition du génome de C. glabrata existants. De nouvelles cassettes intégratives de faible taille et positivement sélectionnables ont été mises au point. Ces éléments présentent toutes les caractéristiques permettant leur utilisation dans la modification du génome de souches sauvages et d’isolats cliniques de C. glabrata. / Candida glabrata is an opportunistic pathogenic yeast, and is today the second causative agent of candidemia in Europe and North America. This yeast has many genomic peculiarities such as the presence of new structural domains within ubiquitous non-coding RNAs. The first aspect of this thesis was the study of the atypical RNA subunit of the nuclear Ribonuclease P of C. glabrata. This RNA contains three large additional domains giving the transcript an overall size more than three times larger than the average eukaryotic RNase P RNA subunits. The experiments performed led to a better understanding of the role of these additional domains and demonstrated for the first time the presence of the Rcl1 protein within the RNase P complex. Secondly, this thesis work also contributed to the improvement of existing genome editing tools in C. glabrata. New small and positively selectable integrative cassettes have been developed. These elements exhibited all the required characteristics for their use in wild-type strains and clinical isolates of C. glabrata.