Coronary artery disease is the leading cause of death worldwide and results from progression of atherosclerosis, which is triggered in part by elevated plasma concentrations of LDL cholesterol. Genome-wide association studies have identified many loci that are associated with circulating lipid levels and bioinformatics tools have been implemented to prioritize positional candidate genes. This project aims to better understand the genetics underlying the regulation of plasma LDL levels and their effect of atherosclerosis using a zebrafish (Danio rerio) model system. A multiplex line with the genes abcg5, abcg8, myrf, col4a3bpa, col4a3bpb, st3gal3, ywhaqa and ywhaqb targeted by CRISPR/Cas9 technique was established using zebrafish with fluorescently labeled macrophages (Tg[mpeg1:mCherry]) and neutrophils (Tg[mpo:EGFP]). Monodansylpentane cadaverase was used to visualize lipids droplets, together with macrophages and neutrophils, in 384 overfed larvae, allowing the visualization and quantification of vascular atherogenic traits at 10 days post-fertilization. Euthanized larvae were homogenized for the quantification of triglycerides, total cholesterol, LDL, HDL, glucose and protein levels. DNA was extracted and larvae were paired-end sequenced for the CRISPR-targeted sites. Linear regression analysis to compare the wild-type larvae against homozygous mutants and additive models for orthologous genes were performed. The lower accumulation of lipids and the lower co-localization of macrophages and neutrophils in the vasculature suggested that the larvae with mutations in the gene abcg5, abcg8, col4a3bpb, and ywhaqb resulted in larvae more protected against atherosclerotic phenotype. The study suggested that loss of function of the targeted genes was associated with atherogenic traits, helping to understand the pathophysiology of atherosclerosis.

The ability to precisely modify the mouse genome is an invaluable tool for any researcher. If an artificial epitope sequence is integrated into target loci in specific cell types, it is possible to generate mice with these cells specifically tagged with the epitope, which can be used for many subsequent studies. Homologous recombination and the Cre/loxP system have been used to generate targeted and conditional transgenic mice, which have provided the basis for many studies into gene function. However, in recent years, improvements in technology have led to the development of RNA and protein based methods of specifically editing DNA sequences at user-selected loci. This thesis aimed to investigate the effectiveness of the novel gene targeting methods TALEN and CRISPR/Cas9. It also aimed to utilize different strains of mice generated using the Cre/loxP system in Trichuris muris, an animal infection model of the human disease Trichuris trichiura. TALENs use a pair of protein-based monomers specific to the sense and anti-sense strand of a target DNA sequence to dimerise a FokI nuclease and initiate a deletion in the genome. As a study into the practical use of this emerging technology TALENs were generated to target Oct-4 (a stem cell marker) in order to integrate an artificial epitope sequence, which could be used for enrichment experiments. The CRISPR/Cas9 is a very efficient RNA-based system used for modifying a target sequence. This system has been utilized to integrate an epitope sequence into the Rosa26 locus, downstream of a floxed STOP codon. This allows for expression of the epitope following the introduction of tissue restricted Cre recombinase. IL-1 is an important cytokine in the immune response towards T.muris. IL-1R1 was conditionally removed in CD4 cells and the role of IL-1 signaling in developing Th1, Th2 and Th17 responses was then studied. Interestingly, IL-1R1fl/fl CD4Cre mice could generate Th1 and Th2 response but showed a reduction in IL-22 and IL-17 production, two key Th17 cytokines. Infected IL-1R1fl/fl CD4Cre mice also displayed increased gut morphology and goblet cell hyperplasia. Therefore, it was concluded that IL-1 signaling from CD4 cells has an important role in host defense and the development of a full Th17 response. It was also shown that removing IL-1R1 in naïve mice had no affect on lymphocyte development. IL-10 is an anti-inflammatory cytokine expressed by gut macrophages, which contributes to homeostatic control of the immune system. IL-10R was specifically removed in the macrophage specific Cre lines LysMCre and also in CX3CR1Cre as a way of comparing the two Cre drivers. The mice were then infected with T.muris and displayed significant inflammation and also failure to expel the worms in the LysMCre model. This suggests a role for this model in future studies of gut macrophages. Clearly, animal models are very important in the study of gene function and also as a method of assessing the application of new technologies such as CRISPR/Cas9. Future work with the artificial epitope specifically targeted into important cell lines will form the basis of many important studies directly applicable to human disorders. As the technologies improve, the scope for developing therapeutics increases and genetic modification has an immeasurable role to play.

There is an unmet need to develop and test treatments for rare lysosomal disease (LD). Most LDs are present in childhood and do not currently have approved therapies. Rare diseases individually are uncommon but taken together account for a population prevalence of 3.5-5.9% worldwide. Due to their rarity, it often takes significant time and effort to diagnose rare diseases. New diagnostic tools, especially for early detection, will offer an advantage in avoiding this diagnostic odyssey. This dissertation is focused on investigating novel diagnostic and treatment methods in vitro for two neurodegenerative LDs: Gaucher disease (GD) and mucopolysaccharidosis IIIB (MPS IIIB). Mutations in NAGLU and GBA1, the genes that encode for lysosomal hydrolases required for degradation of heparan sulfate and glucocerebrosides, lead to the observed pathogenesis in MPS IIIB and GD, respectively. Since many LDs, including MPS IIIB and some forms of GD, are neurodegenerative, cell and gene-based therapeutic strategies are of significant interest. Therapeutics that offer some symptom mitigation in other LDs, such as enzyme replacement or substrate reduction therapies, do not offer appreciable disease mitigation in MPS IIIB or neurodegenerative GD. Here, a novel compound heterozygous mutation, NAGLUY140C/R297X, that results in approximately 50% residual NAGLU protein and 0.6% NAGLU enzyme activity is reported in NAGLU. Furthermore, a RFLP and site-directed mutagenesis strategy was developed to identify the presence of the relatively common p.R297X mutation in patient cell samples, in addition to two other novel molecular assays for the detection of the p.E153K mutation in NAGLU and p.N370S mutation in GBA1. MPS IIIB and GD human skin fibroblasts were reprogrammed to iPSCs using non-integrating Sendai viral vectors with a reprogramming efficiency of 0.2% and 0.3%, respectively. Resulting iPS cell lines were confirmed as being pluripotent through a barrage of analyses for markers of pluripotency and differentiation. Intriguingly, early passage MPS IIIB iPSCs were found to exhibit increased cell death and spontaneous differentiation to embryoid body-like structures, which was hypothesized to be caused by fibroblast growth factor 2 (FGF2) sequestration or degradation due to inherent heparan sulfate dysregulation. Supplemental FGF2 (100 ng/mL) was found to significantly increase confluency of MPS IIIB iPSCs after 48 hours (n = 5, p ≤ 0.05) and persisting to 96 hrs (n = 5, p ≤ 0.05), thus providing evidence for an important role of FGF2-heparan sulfate interactions in the maintenance of stem cell pluripotency. These findings highlight the importance of considering inherent disease pathology when developing disease models. Three genome editing strategies, CRISPR-Cas9, base and prime editing, are addressed throughout this dissertation. Genome editing outcomes in NAGLU and GBA1, as well as a control gene, HPRT1, are reported in HEK293 cells, human skin fibroblasts, and induced pluripotent stem cells (iPSCs). Although CRISPR-HDR failed to yield mutation correction, base editing of the common p.N370S (c.1226 A>G) in GD skin fibroblasts using with 42% efficiency is reported. Base editing of HPRT1 in HEK293 cells with an overall editing efficiency of 6 ± 0.5% (n = 3), but interestingly, when base editing at the centered nucleotide was analyzed, the editing efficiency increases to 27 ± 4.3% (n = 3). These findings align with other reports of a centered nucleotide preference for base editors and will help direct genome editing strategies in the future. This dissertation describes the first genome editing in NAGLU, and the first base editing in GBA1, and underscores the importance of optimizing genome editing strategies when targeting disease-causing mutations in patient-derived cells. The findings reported here will direct future genome editing strategies for developing cell and gene-based therapies for MPS IIIB and GD. / Graduate / 2021-12-15

abstract: Alzheimer’s disease (AD) affects over 5 million individuals each year in the United States. Furthermore, most cases of AD are sporadic, making it extremely difficult to model and study in vitro. CRISPR/Cas9 and base editing technologies have been of recent interest because of their ability to create single nucleotide edits at nearly any genomic sequence using a Cas9 protein and a guide RNA (sgRNA). Currently, there is no available phenotype to differentiate edited cells from unedited cells. Past research has employed fluorescent proteins bound to Cas9 proteins to attempt to enrich for edited cells, however, these methods are only reporters of transfection (RoT) and are no indicative of actual base-editing occurring. Thus, this study proposes a transient reporter for editing enrichment (TREE) and Cas9-mediated adenosine TREE (CasMasTREE) which use plasmids to co-transfect with CRISPR/Cas9 technologies to serve as an indicator of base-editing. Specifically, TREE features a blue fluorescent protein (BFP) mutant that, upon a C-T conversion, changes the emission spectrum to a green fluorescent protein (GFP). CasMasTREE features a mCherry and GFP protein separated by a stop codon which can be negated using an A-G conversion. By employing a sgRNA that targets one of the TREE plasmids and at least one genomic site, cells can be sorted for GFP(+) cells. Using these methods, base-edited isogenic hiPSC line generation using TREE (BIG-TREE) was created to generate isogenic hiPSC lines with AD-relevant edits. For example, BIG-TREE demonstrates the capability of converting Apolipoprotein E (APOE), a gene associated with AD-risk development, wildtype (3/3) into another isoform, APOE2/2, to create isogenic hiPSC lines. The capabilities of TREE are vast and can be applied to generate various models of diseases with specific genomic edits. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22699号 / 医科博第114号 / 新制||医科||8(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 影山 龍一郎, 教授 妻木 範行, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Many of the cutting-edge innovations on the CRISPR/Cas9 system involve the use of complex sgRNA libraries. For example, such libraries have recently been used in functional genomics applications to screen for specific genes in both cell lines and living organisms, as well as in subcellular imaging to fluorescently label chromatin in living cells. However, chemically synthesizing customized libraries can be cost prohibitive, requires precise genomic information, and can takes several weeks. We developed a rapid and efficient enzymatic method to generate sgRNA libraries from arbitrary DNA substrates, significantly reducing the cost of library generation. A type IIS restriction enzyme binding site was incorporated directly into the sgRNA scaffold sequence without affecting the catalytic properties of the Cas9 complex, resulting in a simple method that generates high fidelity sgRNA libraries. Additionally, the library is constructed on the surface of streptavidin coated magnetic beads and a strand displacing polymerase is used to elute DNA from the beads, reducing the loss of material and simplifying purification between steps. We used this method to generate two sgRNA libraries. The first targets the entire E. coli genome, and the second targets genes expressed in the developing zebrafish heart. This second library will be used in a forward genetic screen in zebrafish to identify genes underlying heart defects. These complex sgRNA libraries show the utility of the method in generating libraries from DNA from any species, ranging from prokaryotes like bacteria, to eukaryotes including plants and animals.

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.

Bacterial species and communities play foundational roles in human health and therapeutics, in vital ecological and environmental processes, and in industrial applications for the biosynthesis of valuable compounds and materials. However, existing genetic engineering methods and technologies available for bacterial functional genetics or large-scale genomic integration are inefficient, unable to translate between different target species, or lacking precise targeting or reprogramming capabilities. In this work, we describe a novel class of CRISPR- associated transposons (CRISPR-Tn) that facilitate programmable RNA-guided DNA insertions. In particular, the Tn6677 CRISPR-Tn system from Vibrio cholerae comprises a Tn7-like transposase machinery that has co-opted a nuclease-deficient Type I-F3 CRISPR-Cas system to guide its target selection. We show that, similar to canonical CRISPR-Cas systems, this CRISPR- Tn system can be easily programmed using the CRISPR RNA (crRNA) spacer sequence, and directs highly target-specific DNA integration into the Escherichia coli genome. After defining their core biological and mechanistic principles, we developed these CRISPR-Tn systems into a genome engineering platform, which we named INTEGRATE (Insertions of Transposable Elements by Guide RNA-Assisted Targeting). Particularly, optimization of V. cholerae Tn6677 (Vch INTEGRATE, or VchINT) produced a system capable of programmable, broad-bacterial- host, and multiplexed integration of DNA payloads up to 10 kilobases in length, with genomic editing efficiencies reaching 100%. Our single-plasmid expression of system components enabled, for the first time, genome engineering of specific target strains within a complex fecal bacterial community. In addition, we performed extensive deep sequencing within transposition experiments to characterize and examine non-conventional transposition products, including cointegrates formed through replicative transposition, and long-range integration events resulting from on-target DNA binding. Finally, by individually inserting transposon ends into the E. coli genome, we demonstrated successful transposition-mediated mobilization of a genomic fragment 100 kilobases (kb) in length, demonstrating engineering at the genome-scale using VchINT. Altogether, this work highlights the potential of VchINT and other CRISPR-Tn systems as next- generation genome engineering technologies in bacteria and beyond.

abstract: Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies. / Dissertation/Thesis / Masters Thesis Biology 2020