>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method.

The development of a drug able to distinguish between tumor and host cells has been long sought, but the solid tumor microenvironment (TME) confounds many current therapeutics. Solid tumors present several challenges for oncotherapeutics, primarily, (1) aberrant vascularization, resulting in hypoxia, necrosis, abnormally high pH, and (2) tumor immune suppression. Oncolytic microbes are drawn to this microenvironment by an innate ability to selectively penetrate, colonize, and eradicate solid tumors as well as reactivate tumor associated immune components. To consider oncolytic bacteria deployment into this microenvironment, Chapter 1 dives into the background of oncolytic microbes. A discussion of the oncolytic bacterial field state, identifying Clostridium novyi? as a promising species, and details genetic engineering techniques to develop customized bacteria. Despite the promise of C.novyi in preclinical/clinical trials when administered intratumorally, the genetic and biochemical uniqueness of C.novyi necessitated the development of new methodologies to facilitate more widespread acceptance. Chapter 2 reports the development of methods that facilitate experimental work and therapeutic translation of C.novyi, including the ability to work with this obligate micro-anaerobe aerobically on the benchtop. While methods development is a necessary step in the clinical translation of C.novyi so too is choosing the correct model of the TME within which to test a potential anti-cancer therapy. While the typical solid TME includes both phenotypic and genotypic heterogeneity, the methods used to model this disease state often do not reflect this complexity. This simplistic approach may have contributed to stagnant five-year survival rates over the past four decades. Nevertheless, simplistic models are a necessary first step in clinical translation. Chapter 3 explores the impact of cancer cell lines co-cultured with C. novyi to establish the efficacy of this oncolytic bacteria in a monolayer culture. Chapter 4 extends this analysis adding not only a level of complexity by using an in vivo model, but also using CRISPR/Cas9 to modify the genome of C.novyi to encode a tumor targeting peptide, RGD, for expression within the spore coat. The combination of these studies indicates that C. novyi is uniquely poised to accomplish the long sought after selective tumor localization via intravenous delivery.

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with a poor prognosis. Currently, prognosis for HCC patients remains poor as few therapies are available. The clinical need for more effective HCC treatments remains unmet partially because HCC is genetically heterogeneous and HCC driver genes amenable to targeted therapy are largely unknown. Mutations in the TP53 gene are found in ~30% of HCC patients and confer poor prognosis to patients. Identifying genes whose depletion can inhibit HCC growth, and determining the mechanisms involved, will aid the development of targeted therapies for HCC patients. Therefore, the first half of this thesis focuses on identifying genes that are required for cell growth in HCC independent of p53 status. We performed a kinome-wide CRISPR screen to identify genes required for cell growth in three HCC cell lines: HepG2 (p53 wild-type), Huh7 (p53-mutant) and Hep3B (p53-null) cells. The kinome screen identified 31 genes that were required for cell growth in 3 HCC cell lines independent of TP53 status. Among the 31 genes, 8 genes were highly expressed in HCC compared to normal tissue and increased expression was associated with poor survival in HCC patients. We focused on TRRAP, a co-factor for histone acetyltransferases. TRRAP function has not been previously characterized in HCC. CRISPR/Cas9 mediated depletion of TRRAP reduced cell growth and colony formation in all three cell lines. Moreover, depletion of TRRAP reduced its histone acetyltransferase co-factors KAT2A and KAT5 at the protein level with no change at the mRNA level. I found that depletion of KAT5, but not KAT2A, reduced cell growth. Notably, inhibition of proteasome- and lysosome-mediated degradation failed to rescue protein levels of KAT2A and KAT5 in the absence of TRRAP. Moreover, tumor initiation in an HCC mouse model failed after CRISPR/Cas9 depletion of TRRAP due to clearance via macrophages and HCC cells depleted of TRRAP and KAT5 failed to grow as subcutaneous xenografts in vivo. RNA-seq and bioinformatic analysis of HCC patient samples revealed that TRRAP positively regulates expression of genes that are involved in mitotic progression. In HCC, this subset of genes is clinically relevant as they are overexpressed compared to normal tissue and high expression confers poor survival to patients. I identified TOP2A as one of the mitotic gene targets of the TRRAP/KAT5 complex whose inhibition greatly reduces proliferation of HCC cells. Given that this was the first time the TRRAP/KAT5 complex has been identified as a therapeutic target in HCC, the second half of this thesis focuses on identifying the mechanism via which depletion of this complex inhibits proliferation of HCC cells. I discovered that depletion of TRRAP, KAT5 and TOP2A reduced proliferation of HCC cells by inducing senescence. Typically, senescence is an irreversible state of cell cycle arrest at G1 that is due to activation of p53/p21 expression, phosphorylation of RB, and DNA damage. Surprisingly, induction of senescence after loss of TRRAP, KAT5 and TOP2A arrested cells during G2/M and senescence was independent of p53, p21, RB and DNA damage. In summary, this thesis identifies TRRAP as a potential oncogene in HCC. I identified a network of genes regulated by TRRAP and its-cofactor KAT5 that promote mitotic progression. Moreover, I demonstrated that disruption of TRRAP/KAT5 and its downstream target gene TOP2A result in senescence of HCC cells independent of p53 status. Taken together, this work suggests that targeting the TRRAP/KAT5 complex and its network of target genes is a potential therapeutic strategy for HCC patients.

Permanent repair of the F9 gene is a significant goal to cure Hemophilia B disease. Advanced gene therapy using CRISPR/Cas9 system can increase circulation level of Factor IX proteins to a significant level without the need of demanding infusions of FIX concentrates. Induced pluripotent stem cells represent an ideal cell for gene therapy because patient-derived cells could be reprogrammed into iPSCs, genetically modified, selected, expanded and then induced to differentiate into fully functional hepatocytes in vitro. This study covered a portion of a 5-year project which ultimately aims at establishing therapeutic results in transgenic Hemophilia B mice by injecting genetically corrected iPSC-derived hepatocytes into the liver. The purpose of this thesis is to summarize what has been completed up to now: generation of the proper model of Hemophilia B human iPSCs using CRISPR/Cas9-mediated genome editing and differentiation of healthy and disease specific iPSCs into hepatocytes which will allow disease modelling to look for cell function, viability, homogeneity and drug screening. Further research will be done to effectively knock-in the F9 allele into liver safe harbor site of disease specific iPSCs, which will express FIX at a significant level to show therapeutic effects.

Rare gene-disrupting variants and common regulatory variants play key roles in rare and common disease, respectively. These variants are of great interest for investigation into genetic contributions to disease, but experimental methods to validate their impact on gene expression levels are lacking. In this study, we utilized CRISPR/Cas9 genome editing to validate regulatory variants including cis-eQTLs, rare stop-gained variants in healthy and disease cases and one immune-response trans-eQTL master regulator. For investigation into common and rare regulatory variants within transcribed regions, we developed a scalable CRISPR-based polyclonal assay for experimental assessment. First, we applied this assay to nine rare stop-gained variants found in the general population, in GTEx. After editing, the stop-gained variants show a significant allele-specific depletion in transcript abundance, as expected. Next, we utilized the assay to validate 33 common eQTLs found in GTEx. After editing, the eQTL variants show higher variance in effect size than control variants, indicating a regulatory effect. Finally, we applied the polyclonal editing approach to clinical and new stop-gained variants in two disease-associated genes. The results follow the expected trend, with NMD being triggered by variants upstream of the NMD threshold but not by those beyond. This method demonstrates scalable experimental confirmation of putative causal regulatory variants, and improved interpretation of regulatory variation in humans. Next, we sought to experimentally validate an immune-response eQTL for IRF1 in cis and many genes in trans under LPS stimulation. We used CRISPRi to repress the enhancer locus and found that the enhancer is active in our immune cell system. Next, we used CRISPR-Cas9 genome editing and isolation of monoclonal cell lines to target this variant locus. After LPS stimulation, we performed RNA-sequencing on wild type and edited clones, showing that the effect size of the genes which are associated with the trans-eQTL are correlated with differential expression between the edited and wild type cell lines for the same genes. Additionally, we find that the differential expression between edited clones is correlated with CRISPRi repression of the IRF1 promoter and enhancer. In this way, we were able to identify a common genetic variant which modifies the transcriptomic immune response to LPS and validate the trans-eQTL signal.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第17893号 / 工博第3802号 / 新制||工||1581(附属図書館) / 30713 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19765号 / 農博第2161号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4981(農学部図書室) / 32801 / 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, 教授 澤山 茂樹, 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

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21689号 / 医科博第93号 / 新制||医||1036(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 斎藤 通紀, 教授 中川 一路, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Clustered regularly interspaced short palindromic repeats (CRISPR) are derived from a bacterial and archaeal adaptive immune system. The core enzymes of CRISPR are RNA-guided endonucleases that sequence-specifically cleave foreign double-stranded DNA. Improving and controling the properties of the CRISPR system is a crucial step in advancing the therapeutic potential of CRISPR technology. Several classes of these enzymes exist and are being adapted for biotechnology, such as genome engineering. Cas12a (Cpf1) is a Type V CRISPR-associated (Cas) enzyme that naturally uses only one guide RNA, in contrast to Type II CRISPR-Cas9 enzymes. Thus, Cpf1 may represent a simpler, more practical tool for applications such as gene editing and therapeutics. This dissertation comprises four related studies in this area. To better understand the functional requirements for Cpf1-crRNA interaction and develop modified crRNAs suitable for synthetic biology and therapeutic applications, the first study performed nucleotide substitutions in the crRNA. It focused on the protein-interaction motif of the crRNA by incorporating base changes at the 2ʹ position that alter hydrogen-bonding capacity, sugar pucker, and flexibility. DNA substitutions in RNA can probe the importance of A-form structure, 2ʹ-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. In addition, Chemical modifications include 2'-deoxy, 2'-fluoro, 2'- deoxy-arabinonucleic acid, and oxepane. Our study discovered that 2'-fluoro maintains the A-form structure and is compatible with AsCpf1 activity. Biochemical endonuclease activity, gene editing efficiency, Cpf1 binding affinity, and ribonucleoprotein stability were used to assess the tolerance and effects of modification. Characterizing structure-function requirements for Cpf1-crRNA interaction will facilitate better design and tuning of Cpf1 enzymes. The second study established a FRET-based assay in collaboration with a computational collaborator to identify small molecule inhibitors predicted by virtual docking and simulations. This study aims to lay the foundation for efficient, safe implementation of CRISPR-Cpf1. The third study used chemically modified Cas9-guide RNAs to offset known weaknesses of CRISPRi. It takes advantage of the high binding affinity and nuclease resistance of modified guides to potentially reduce the required components for CRISPRi.