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The cardiovascular system is an essential component of mammalian biology. It is a complex network of various tissues and structures with unique functions. The function of the cardiovascular system is to supply nutrients including oxygen to the various cells, tissues and organs within the body, and remove waste products from them. Given the importance of this role, it is not surprising that there are countless regulatory mechanisms at the molecular, cellular and tissue levels that are required to support this functional system. Perturbations in parts of this system are likely to lead to abnormalities, and thus give rise to cardiovascular-related diseases. Despite the currently expanding list of genes reported to be involved in a variety of cardiovascular-related diseases, including calcific aortic valve disease (CAVD), the functions and associated pathways of these factors in both normal and pathological physiology have yet to be fully understood, such as at the transcriptomic level. In this thesis, a genome-wide transcriptomic atlas of the healthy mammalian cardiovascular system was generated using the sheep as a large animal model. This atlas was generated using RNA-seq, with the aim of further understanding normal gene expression patterns in the context of the known physiology of healthy mammalian tissues. Through this work, I identified novel gene networks and detailed functional clustering of co-expressed genes with region-specific expression and specialised cardiovascular roles. One interesting cluster was highly expressed in the cardiac valves, and shared genes found in physiological bone development, such as bone morphogenetic protein 4 (BMP4), collagen type I alpha 2 (COL1A2), Sry homeobox 8 (SOX8) and bone gamma-carboxyglutamate protein (BGLAP), some of which have been implicated in vascular calcification. Further to this work, I studied the expression profiles of these key cardiovascular genes during development in the sheep from foetal to adult stages. In addition, I investigated the gene expression patterns of various key vascular calcification genes. These studies showed differential expression of genes in the different cardiovascular tissues, demonstrating transcriptional differences between these different tissues known to have different functions. CAVD involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine the mechanisms of CAVD. However, to date, no published studies have utilised cell lines to investigate this process Thus, in this project, I generated and evaluated the calcification potential of an immortalised cell line derived from sheep aortic VICs (SAVICs). This novel large animal in vitro model of CAVD was demonstrated to calcify under high calcium and phosphate conditions. Changes in the expression of key calcification genes during VIC calcification was also observed, including increased mRNA expression of bone markers Runt-related transcription factor 2 (RUNX2) and sodium-dependent phosphate transporter 1 (PiT1), and a concomitant decrease in matrix Gla protein (MGP) mRNA expression. In addition, the role of extracellular nucleotides and their receptors (P2 receptors), which have been previously shown to be important in bone and vascular calcification, were investigated using SAVICs in vitro. This study has shown that extracellular nucleotides, particularly adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP) and other agonists of P2 receptors, reduced VIC calcification in vitro. Moreover, the cutting-edge gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), was successfully applied to generate large animal models of cardiovascular-related diseases. In this project, I applied the CRISPR/Cas9 technology to edit ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and fibrillin 1 (FBN1) to generate two models of vascular calcification and Marfan Syndrome (MFS), respectively. In the ENPP1-edited animals, soft tissue calcification has been observed in the biallelic mutant and homozygous pigs. In this project, I have developed a range of novel in vitro and in vivo tools to advance the study of cardiovascular disease. These studies demonstrate that large animal models are highly valuable in the field of cardiovascular biology. The in vivo and in vitro experimental models described should facilitate detailed analysis of cardiovascular molecular biology and ultimately lead to therapies which will minimise the morbidity and mortality currently arising from cardiovascular pathology.

With advancements in CRISPR-cas9 broadening the potential paths for clinical usage of genetic editing, conversations about genetic editing have grown to outside simply scientific communities and into mainstream conversations. This study focuses specifically on Christian discourse of genetic editing and locates four major tensions for many Christians when they think about genetic editing: beginning of life, Creator-human relationship, imago Dei, and stewardship. With these major concerns in mind, I identify epigenetics, somatic cell genetic editing, and in vivo genetic editing research as important research paths to pursue as they can potentially produce techniques that more Christian individuals would feel comfortable using. I pursue one of these paths and conclude with an experimental proposal for an analysis of in vivo and in vitro CIRSPR-Cas9 efficiency in regards to on- and off-target rates.

abstract: A novel clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool for simultaneous gene editing and regulation was designed and tested. This study used the CRISPR-associated protein 9 (Cas9) endonuclease in complex with a 14-nucleotide (nt) guide RNA (gRNA) to repress a gene of interest using the Krüppel associated box (KRAB) domain, while also performing a separate gene modification using a 20-nt gRNA targeted to a reporter vector. DNA Ligase IV (LIGIV) was chosen as the target for gene repression, given its role in nonhomologous end joining, a common DNA repair process that competes with the more precise homology-directed repair (HDR). To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression. This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2018

The CRISPR-Cas9 gene editing system gives researchers the ability to manipulate and edit DNA with unprecedented ease and precision. It was discovered in bacteria as part of their adaptive immune system, but has been reengineered to target any double stranded DNA. This burgeoning molecular tool has created great excitement as scientists are rapidly adopting it to study fields including human gene therapy, disease modeling, agriculture, gene drive in mosquitos, and many others. This paper will explore the potential impact of CRISPR-Cas9 in human therapeutics. Specifically, the potential of CRISPR-Cas9 to treat Duchenne Muscular Dystrophy will be examined. In several ways, this debilitating degenerative disease is an ideal candidate for gene-editing with CRISPR-Cas9. Recent progress in the lab has demonstrated the gene editing system’s ability to rescue dystrophin protein levels in vivo. Although CRISPR-Cas9 holds great promise for previously incurable diseases, there are still many limitations that must be overcome before the gene editing system can be used in patients. This paper will discuss these barriers as well as recent advancements to overcome them.

The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

The discovery and development of clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have revolutionized targeted genomic medicine. In my thesis, we discuss our efforts to improve and optimize various CRISPR-Cas systems for therapeutic genome editing applications. In part one, we propose that aberrant splice site disruption could be a simple and efficient strategy for treating some mutations associated with β-thalassemia. Specifically, we show that disruption of common mutant alleles in the HBB gene by Cas9 and Cas12a results in restoration of normal β-globin splicing, functional expression of HBB, and improved quality of erythroid maturation in edited β-thalassemia patient CD34+ hematopoietic stem and progenitor cells. In part two, we demonstrate that optimization of the nuclear localization signal (NLS) sequence framework is an effective method to improve the mutagenesis frequencies of Cas12a nuclease. In particular, the 3xNLS-NLP-cMyc-cMyc framework improves genome editing in mammalian and primary cells, relative to previous Cas12a NLS frameworks. We show that our NLS optimization approach can be applied to various Cas12a orthologs resulting in high editing activity without sacrificing the high intrinsic specificity of Cas12a nucleases. Furthermore, we demonstrate that NLS-optimized enAspCas12a can efficiently disrupt the ATF4-binding motif at the +55 enhancer of BCL11A, which may serve as an alternative therapeutic strategy for β-hemoglobinopathies. In part three, we develop and characterize fusion enhanced base editor (feBE) systems, which are fusions of base editors to our Cas9-programmable DNA binding domain (pDBD) and Cas9-Cas9 platforms. We report that our feBEs are more active than previously described base editor platforms at canonical and noncanonical PAMs. Furthermore, we show that feBEs can selectively edit a therapeutically relevant target site, CCR5, with minimal editing at a highly homologous off-target site. Taken together, my thesis research aimed to engineer CRISPR-Cas tools to improve their efficiency and specificity for therapeutic genome editing applications.

Clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) constitute a bacterial and archaeal adaptive immune system. The ongoing arms race between prokaryotic hosts and their invaders such as phages led to the emergence of anti-CRISPR proteins as countermeasures against the potent antiviral defense. Since the first examples of anti-CRISPRs were shown in a subset of CRISPR-Cas systems, we endeavored to uncover these naturally-occurring inhibitors that inactivate different types of CRISPR-Cas systems. In the first part of my thesis, we have identified and characterized Type II anti-CRISPR proteins that inactivate several Cas9 orthologs. We share mechanistic insights into anti-CRISPR inhibition and show evidence of its potential utility as an off-switch for Cas9-mediated mammalian genome editing. Although the RNA programmability of Cas9 enables facile genetic manipulation with great potential for biotechnology and therapeutics, limitations and safety issues remain. The advent of anti-CRISPR proteins presents opportunities to exploit the inhibitors to exert temporal, conditional, or spatial control over CRISPR. In the second part of my thesis, we demonstrate that anti-CRISPR proteins can serve as useful tools for Cas9 genome editing. In particular, we have demonstrated that anti-CRISPRs are effective as genome editing off-switches in the tissues of adult mammals, and we further engineered anti-CRISPR proteins to achieve tissue-specific editing in vivo. Taken together, my thesis research aimed to mine for natural anti-CRISPR protein inhibitors and repurpose these proteins to complement current Cas9 technologies in basic and clinical research.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20930号 / 理博第4382号 / 新制||理||1630(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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