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.

Wnt signaling is an evolutionarily conserved pathway that is essential for the development of the metazoan embryo. Wnt signaling controls essential developmental processes including cell fate, cell polarity, dorsal-ventral patterning and tissue movement. Misregulated Wnt signaling can have disastrous effects on the developing human embryo, leading to potentially fatal congenital malformations including anencephaly and spina bifida. In addition to embryonic development, misregulated Wnt signaling has been implicated in human pathologies including colon and breast cancers and skeletal malformations. Wnt signaling is divided into two main pathway branches, canonical or beta-catenin dependent, and non-canonical, sometimes referred to as the planar cell polarity (PCP) pathway. The former branch activates the transcription of the downstream target genes leading to the patterning the dorsal-ventral axis of the developing embryo, whilst the latter has no downstream transcriptional targets but rather acts upon the cytoskeleton to control cell and tissue polarity and movement. Wnt signaling bifurcates into these two branches at the level of the protein Dishevelled (Dvl). The Dishevelled-associated activator of morphogenesis 1 (Daam1) protein was identified via a yeast-two hybrid screen using Dvl as bait. Daam1 interacts directly with Dvl and mediates activation of the small GTPase Rho, a key player in non-canonical Wnt signaling necessary for proper gastrulation in the Xenopus (frog) embryo. In addition to Daam1, vertebrates possess a second Daam, Daam2, originally identified via an in silico screen in humans. Similar to Daam1, frog Daam2 participates in non-canonical Wnt signaling, contributing to proper formation of the embryonic neural tube. However, conflicting opinions on the function of Daam2 have led to discrepancies regarding its position in Wnt signaling and function in development. Daam1/2 have not been extensively investigated at the genetic level, therefore, I employed the genetic model zebrafish (Danio rerio) to further clarify their role in Wnt signaling. Using techniques such as the latest gene-editing system CRISPR/Cas9 and other well-established molecular methods including in situ hybridization, RT-PCR and knockdown using morpholino oligonucleotides, I sought to further establish the role of the Daam protein family in vertebrate embryonic development. Together, my results indicate that the zebrafish Daam1a/b and Daam2 behave similarly to Daam1 and Daam2 in frog, respectively, by participating in the non- canonical Wnt signaling pathway and mediating morphology in the developing zebrafish embryo. / Biology

Selenoprotein H (SELENOH) is a nucleolar oxidoreductase with DNA binding properties whose function is not well understood. To determine the functional and physiological roles of SELENOH, a knockout of SELENOH was generated in cell lines using CRISPR/Cas9-mediated genomic deletion and in mice by targeted disruption. Based on the sequenced genome, the results of deduced protein sequences indicated various forms of mutants in the CRISPR/Cas9-mediated knockout, including a frame-shift by aberrant splicing and truncated SELENOH by early termination of the translation process. Loss of SELENOH in HeLa cells induced slow cell proliferation, the formation of giant multinucleated cells, accumulation of unrepaired DNA damage and oxidative stress, and cellular senescence. SELENOH cells were enlarged and possessed a single large nucleolus. Atomic force microscope showed increased stiffness in the nucleoli of SELENOH knockout cells, which suggests that SELENOH maintains the flexible structure of the nucleolus. Furthermore, the knockout of SELENOH led to a large-scale reorganization of the nucleolar architecture with the movement of nucleolar protein into nucleolar cap regions in response to oxidative stress. The nucleolar reorganization is dependent on ATM signaling. Altogether, results suggest that SELENOH appears to be a sensor of oxidative stress that plays critical roles in redox regulation and genome maintenance within the nucleolus. To determine the physiological role of SELENOH in vivo, Selenoh knockout mice were generated by targeted deletion through homologous recombination. Selenoh+/− mice were fertile and phenotypically indistinguishable from wild-type littermates. Results from matings of Selenoh+/− mice showed a significantly reduced fraction of Selenoh−/− offspring on the basis of Mendelian segregation. Since some Selenoh−/− were born, it is likely that Selenoh is a partially essential gene in mice. Live-born Selenoh−/− mice were viable and born without apparent phenotypes. Selenoh−/− mice at 2-month of age showed increased GPX activity in the lung but not in the brain and liver. Furthermore, loss of Selenoh resulted in the aggravated formation of aberrant crypt foci in the colon of Selenoh+/− mice that were injected with azoxymethane. Altogether, SELENOH has critical roles in embryogenesis and colorectal carcinogenesis.

NK cell based anti-tumour therapies demonstrate high efficacy in targeting hematological malignancies, however, treatments for advanced solid tumours face challenges. The immunosuppressive environment produced by tumours prevents NK cells from maintaining cytotoxic activity and reducing tumour burden. Enhancing NK cell activation is essential to improve their function against solid tumours. Genetic manipulation of primary NK cells with viral and non-viral methods has seen a drastic improvement in recent years. Lentiviral vectors are being used to generate CAR-NK cells ex vivo, while refinement of electroporation protocols has allowed for the generation of stable gene knockouts in primary NK cells. To establish and validate the generation of a stable knockout in primary human NK cells we focused on targeting the NCAM-1 (CD56) surface adhesion molecule. The high surface expression of CD56 in NK cells makes it a suitable target to establish the knockout protocol. Furthermore, despite its levels of expression being correlated to different functional phenotypes, the role of CD56 in NK cell function is not understood. Here we have shown that current lentiviral transduction protocols are not viable methods to deliver the sgRNA/Cas9 system into primary NK cells. However, we found that nucleofection of the sgRNA/Cas9 complex into NK cells is an efficient method to generate gene knockouts. Using newly generated CD56KO NK cells we have shown that the expression of CD56 has no effect on NK cell cytotoxicity, cytokine production, proliferation, and in vivo tissue trafficking. In parallel, we have also identified an intracellular pathway that is active in the tumour microenvironment and could inhibit NK cell function. Recent studies on the intracellular signaling of the E3 ubiquitin-protein ligase Cbl-b have highlighted its role in inhibiting NK cell tumour lytic and anti-metastatic activity. Immunosuppressive factors produced by tumours activate the Cbl-b pathway, leading to the targeted degradation of signaling proteins required for NK cell activation. We have shown that Cbl-b is upregulated in ex vivo expanded NK cells cultured with GAS6 or ovarian cancer ascites. Therefore, the generation of human primary Cbl-bKO NK cells could be a beneficial asset to enhance NK cell cancer immunotherapy. / Thesis / Master of Science (MSc)

Clustered regularly interspaced palindromic repeats (CRISPR) and their associated (Cas) proteins co-opted as biotechnological tools have improved the simplicity and accessibility of gene editing for fields ranging from crop science to the treatment of human disease. These technologies, however, come with an inherent degree of risk associated with off-target events or direct misuse, accidental or intentional, leading to permanent genetic damage to ecosystems, livestock, or people. Naturally occurring anti-CRISPR proteins have been described, as well as synthetic small molecule inhibitors, but each of these approaches, while suitable for certain applications, leaves something to be desired in deliverability or efficacy in the face of many possible adverse CRISPR-related events. Inspired by strides in the field of oligonucleotide therapeutics, we developed the first reported anti-CRISPR nucleic acids for Streptococcus pyogenes (Sp)Cas9 to address the critical need for fail-safe inhibitors of Cas enzymes. These inhibitors, termed small nucleic acid-based inhibitors of Cas9 (SNuBs), comprise two modules which act in tandem to bind and disable the SpCas9 RNP. We have demonstrated that SNuBs inhibit Cas9 in vitro and in human cells. Successive rounds of optimization on our initial designs have yielded inhibitors capable of carrier-free uptake into human cells, high nuclease resistance, and robust inhibition at low stoichiometric concentrations relative to Cas9 and its RNA guide. In their current form, SNuBs quite possibly present the most tenable approach to inhibiting Cas9 in a variety of contexts including therapeutic applications in the near future.