京都大学 / 新制・課程博士 / 博士(農学) / 甲第23246号 / 農博第2453号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5336(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 白井 理, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

La dystrophie musculaire de Duchenne est une maladie génétique qui résulte de diverses mutations dans le gène DMD, codant pour la protéine dystrophine. 70% des patients ont une délétion d'exons ou de parties d'exons provoquant un changement dans le cadre de lecture, résultant en l'apparition d'un codon stop et en l'absence de la protéine dystrophine. Plusieurs traitements potentiels ont été explorés dans les dernières années pour cette maladie, dont le système CRISPR-Cas9, un outil génétique permettant d'éliminer un segment d'ADN à l'aide de la protéine nucléase Cas9 et de deux guides d'ARN ciblant des séquences précises d'ADN. Le plus grand défi avec l'utilisation de cette technologie est sa livraison in vivo. Les vésicules extracellulaires sont des particules membranaires lipidiques qui jouent un rôle dans la communication intercellulaire et sont retrouvées dans tous les biofluides chez les mammifères. Elles pourraient donc être une alternative intéressante pour la livraison du système CRISPR-Cas9. J'ai participé à des travaux de purification de vésicules extracellulaires de sérum par chromatographie par exclusion de taille. Ces vésicules extracellulaires ont été chargées avec la protéine Cas9 et des guides ARN, puis, des injections intramusculaires ont été effectuées dans le Tibialis anterior de trois lignées de souris (Ai9, RAG-mdx et mdx/hDMD) pour établir l'efficacité du traitement. Les résultats ont montré que les vésicules extracellulaires chargées avec la Cas9 et des guides d'ARN provoquent une édition de l'ADN efficace dans le Tibialis anterior des trois lignées de souris utilisées et la restauration de l'expression de la protéine dystrophine dans les fibres musculaires du Tibialis anterior des souris RAG-mdx, modèle pour la dystrophie musculaire de Duchenne. Le traitement a ensuite été modifié pour permettre le ciblage des vésicules extracellulaires aux organes affectés par la dystrophie musculaire de Duchenne, soit le cœur et les muscles squelettiques. Des peptides de ciblage ont été sélectionnés dans la littérature et insérés dans la membrane des vésicules extracellulaires marquées de façon fluorescente à l'aide d'un segment lipidique stéaryl. Les résultats de l'expérience effectuée avec les vésicules extracellulaires ciblées n'ont pas été concluants en raison d'un marquage mal adapté des vésicules injectées, mais de futures expériences permettront d'élucider leur efficacité. L'ajustement du traitement pour permettre une injection systémique rejoignant le cœur et les muscles squelettiques est indispensable à l'application de celui-ci à la clinique. / Duchenne muscular dystrophy is a genetic disease that affects one in 3500 boys and results from mutations in the DMD gene, which codes for dystrophin protein. 70% of patients have an exon deletion, which results in a shift in the reading frame, the apparition of a stop codon, and the absence of the dystrophin protein. Many different potential treatments have been explored for Duchenne muscular dystrophy, including the CRISPRCas9 system. This technology allows for the modification of genomic DNA through a Cas9 nuclease and two guide RNAs designed to target a specific DNA sequence. The biggest challenge with using the CRISPR system is delivery. The classic vectors for CRISPR, such as AAV, can cause many adverse effects like immunological responses. Extracellular vesicles are membranous particles that play a role in intercellular communication and are found in all mammalian biofluids. They are thus an interesting alternative for the delivery of the Cas9 protein and its guide RNAs. I have participated in a research project aiming to purify serum extracellular vesicles by size-exclusion chromatography and to load them with Cas9 and two guide RNAs. These extracellular vesicles were then injected intramuscularly into the Tibialis anterior muscles of three mouse strains (Ai9, RAG-mdx and mdx/hDMD) to assess treatment efficiency. The injection of Cas9 and guide RNA-loaded extracellular vesicles produced efficient gene editing as well as dystrophin expression restauration. To modify the treatment for systemic injection, targeting peptides were added to EV membrane through a lipid stearyl segment. This was done to target the extracellular vesicles to Duchenne muscular dystrophy-affected organs: heart and skeletal muscles. Results of the targeted-extracellular vesicle experiment were inconclusive, however, with more experiments, the efficacy of the targeting peptides should be determinable. It is essential to adjust this treatment to allow for targeted systemic delivery for it to be applicable to the clinic.

Las CRISPR son unas repeticiones agrupadas regularmente espaciadas presentes en numerosos y diversos grupos de procariotas. Junto con unos genes asociados a las repeticiones (genes cas) forman los sistemas CRISPR-Cas. En trabajos incluidos en esta tesis se predijo que los sistemas CRISPR-Cas podrían proporcionar inmunidad adaptativa específica guiada por las secuencias espaciadoras, lo que tras comprobarse ha conllevado una revolución en aplicaciones biotecnológicas y en el modo de entender la microbiología. Se analizan en mayor detalle la funcionalidad del sistema CRISPR-Cas I-E de Escherichia coli, su diversidad, evolución y posible uso para el tipado de cepas. En relación con la adquisición de espaciadores se incluye un método patentado para detectarlas y la propuesta de un mecanismo de acción. Dado que el sistema CRISPR-Cas I-E se encuentra presente pero reprimido en la mayoría de cepas silvestres de E. coli, se discute qué papel ha podido desarrollar en la especie.

In many organisms, the most fundamental event during embryogenesis is differentiating between germline cells and specialized somatic cells. In C. elegans, PIE-1 functions to protect the germline from somatic differentiation and appears to do so by blocking transcription and by preventing chromatin remodeling in the germline during early embryogenesis. Yet the molecular mechanisms by which PIE-1 specifies germline remain poorly understood. Our work shows that SUMOylation facilitates PIE-1-dependent germline maintenance and specification. In vivo SUMO purification in various CRISPR strains revealed that PIE-1 is SUMOylated at lysine 68 in the germline and that this SUMOylation is essential for forming NuRD complex and preserving HDA-1 activity. Moreover, HDA-1 SUMOylation is dependent on PIE-1 and enhanced by PIE-1 SUMOylation, which is required for protecting germline integrity. Our results suggest the importance of SUMOylation in the germline maintenance and exemplify simultaneous SUMOylation of proteins in the same functional pathway.

With the discovery and development of CRISPR, the technology that might allow us to modify the human germline is at our fingertips, and, consequently, serious practical and ethical consideration is warranted. In the following paper, I examine the ethics of using CRISPR in this way and argue that modifying the human germline for the purpose of preventing serious genetic disease is, in principle, ethically acceptable and ought to be allowed. I present several arguments to this effect including arguments that rely on the principles of beneficence and autonomy. I also examine the larger societal implications of human germline modification. I then respond to six of the most prominent objections that have been raised against CRISPR and germline gene modification before concluding with a brief discussion of the biggest challenge that we face as we move forward with CRISPR, that of limiting the use of this promising and incredibly versatile technology.

One of the most surprising realisations to emerge from metagenomics studies in the early ‘00s was that the population of viruses and phages in nature is about 10 times larger than the population of prokaryotic organisms. Thus, bacteria and archaea are under constant pressure to develop resistance methods against a population of viruses with extremely high turnover and evolution rates, in what has been described as an evolutionary “arms race”. A novel, adaptive and heritable immune system encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral or plasmid sequences which are then used to inactivate the corresponding invader element via an RNA interference mechanism. A number of CRISPR-associated (Cas) protein families are responsible for the maintenance, expansion and function of the CRISPR loci. This system can be classified in a number of types and subtypes that differ widely in their gene composition and mode of action. This thesis describes the biochemical characteristics of CRISPR-mediated defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci transcription and their subsequent maturation into small guide crRNA units by the processing endonuclease of the system (Cas6) is investigated. After this step, different pathways and effector proteins are involved in the recognition and silencing of DNA or RNA exogenous nucleic acids. This thesis reports the identification and purification of a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue of which has been found to recognise and cleave RNA targets in P. furiosus. The recognition and silencing of DNA targets in E. coli has been shown to involve a multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD nuclease. S. solfataricus encodes orthologues for the core proteins of this complex, and the formation and function of an archaeal CASCADE is investigated in this thesis.

<p>Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.</p> / Dissertation

By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer. / October 2016

In prokaryotes, CRISPR/Cas9 system provides molecular immunity to bacteria and archaea against invading phages, conjugative plasmids and nucleic acids. CRISPR/Cas9 system has been adapted for targeted genome editing across diverse eukaryotic species for a variety of applications in basic and applied research. In this dissertation, I propose to adapt the CRISPR/Cas9 system to function as molecular immunity machinery against plant DNA viruses. Therefore, to test whether the CRISPR/Cas9 system is portable to plants, I produced plants stably over-expressing Cas9 and sgRNAs against single or multiple DNA viruses in Nicotiana benthamiana (N. benthamiana) and tomato (Solanum lycopersicum) plants. sgRNAs targeting the Cas9 endonuclease against different coding and non-coding viral sequences were tested in virus- interference experiments. I explored the possibility of generating robust interference against single and multiple DNA viruses. Subsequently, I studied the possibility of virus evasion of the CRISPR/Cas9 machinery and evolution of the virus escapees. Finally, I produced N. benthamaiana and tomato plants stably expressing the CRISPR/Cas9 machinery for developing durable virus resistance. Furthermore, developing effective viral-interference system in plants will help to understand the molecular underpinning of virus biology and host-defense mechanisms against plant viruses. In conclusion, my research project attempted to establish the efficacy and extend the utility of CRISPR/Cas9 system for viral interference in plants which promise exciting applications including producing engineered plants resistant to multiple viral infection.

Integration and expression of exogenous genetic material – in particular, transgenes – into the genomes of model organisms, cell lines or patients is widely used for the creation of genetically modified experimental systems and gene therapy. However, loss of transgene expression due to silencing is still a major hurdle which remains to be overcome. Judicious selection of integration loci can help alleviate the risk of silencing; in recent years the ability to efficiently and specifically target transgene integration has been improved by the advent of site-specific nucleases (SSNs). SSNs can be used to generate double strand breaks (DSBs) in a targeted manner, which increases the efficiency of homologous recombination (HR) mediated transgene integration into predetermined loci. In this work I investigate four human genomic loci for their potential to act as transgene integration sites which will support robust long term expression: the adeno-associated virus (AAV) integration site 1 (AAVS1); the human homologue of the mouse Rosa26 locus (hROSA26); the inosine monophosphate dehydrogenase 2 (IMPDH2) gene and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene. I also investigate the potential of creating a novel drug-selectable transgene integration system at the IMPDH2 locus to allow for rapid and specific selection of correctly inserted transgenes. In addition to their ability to drive targeted transgene integration, SSNs can be harnessed to specifically disrupt gene function through indel formation following erroneous repair of the induced DSB. Using this strategy, I aimed to answer some important biological questions about eukaryotic translation elongation factor 1 alpha (eEF1A); eEF1A is responsible for providing aminoacylated tRNAs to the ribosome during the elongation phase of protein synthesis. Humans and other vertebrates express two isoforms, eEF1A1 and eEF1A2 (encoded by EEF1A1 and EEF1A2 respectively). During development eEF1A1 is replaced by eEF1A2 in some tissues. The reasons for this remain elusive, but one explanation may lie in the moonlighting functions of eEF1A1, which may not be shared by eEF1A2. Additionally, eEF1A2 can act as an oncogene, while there is no evidence that eEF1A1 is overexpressed in tumours. To begin to untangle these issues I targeted EEF1A1 using SSNs with the aim of making a cell line expressing only the eEF1A2 isoform. This work suggests that eEF1A1 may be essential even in the presence of eEF1A2, though further studies will be required to confirm this.