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

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.

CRISPR-Cas9 är ett effektivt genredigeringsverktyg som har upptäckts på senare år. Verktyget härstammar från ett adaptivt immunförsvar hos prokaryoter. Tekniken används för att modifiera DNA hos växter, djur och människor på ett enkelt och billigt sätt. CRISPR-Cas9 har visat sig ha stor potential vid bekämpning av olika sjukdomar däribland cancer som idag är ett globalt hälsoproblem. Inom cancerforskningen ses CRISPR-Cas9 som ett lovande verktyg vid cancerterapi och läkemedelsutveckling. I denna studie sammanställer vi aktuella metoder och användningsområden med CRISPR-Cas9 inom cancerforskning. Dessutom undersöker vi hur denna form av genteknik kan lyftas upp och tillämpas i biologiundervisningen. / CRISPR-Cas9 has recently emerged as an effective genome editing tool. The tool derives from an adaptive immune system in prokaryotes. The technology is used for modification of DNA in plants, animals and humans in a simple and inexpensive way. CRISPR-Cas9 has shown great potential in fighting different diseases like cancer which today is a global health issue. It is seen as a promising tool for cancer research when it comes to cancer therapy and drug development. Here we summarize current methods and applications of CRISPR-Cas9 for cancer research. Furthermore, we explore the possibilities of introducing and applying this kind of genetic engineering in biology teaching. / <p>Framläggning, opponering och respondering skedde skriftligt till följd av covid19.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22335号 / 医博第4576号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 岩田 想, 教授 木村 剛 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23413号 / 医博第4758号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 小柳 義夫, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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 diploma thesis describes the measurement and evaluation of braking traces in cars equipped by system ABS. The result of the work should contribute to improving the quality of photodocumentation of braking traces. To creating a procedure to eliminate as much as possible the loss of information during tracking documentation.

V tomto texte sa zameriame na populárnu koncepciu kolaboratívnej tvorby dokumentov. Predstavíme si myšlienku využitia tohto mechanizmu v rôznych oblastiach rozhodovania, popíšeme si koncept a princíp fungovania. Následne si predstavíme a rozoberieme portály a portletovú technológiu, ich výhody a využitie. Cieľom práce je implementácia kolaboratívneho editora s využitím knižnice pre prácu so zmenami v dokumentoch s perzistentnou a aplikačnou logikou na platforme JEE a vytvorenie jednoduchého portletu pre túto službu.

Diese Arbeit betrachtet das Cograph Editing Problem im Kontext der Rekonstruktion phylogenetischer Bäume. Insbesondere muss dafür ein Cograph rekonstruiert werden, der durch Rauschen verzerrt wurde. Liu et al. zeigten, dass Cograph Editing NP-vollständig ist. Damit ist das Problem für große Graphen wahrscheinlich nicht in realistischer Zeit korrekt lösbar. Diese Arbeit stellt daher eine Greedy-Heuristik für die Lösung des Cograph Editing Problems vor. Dabei werden Editieroperationen als günstig betrachtet, wenn sie die Anzahl an induzierten P4 in dem Graphen minimieren. Es wird ein on-line Algorithmus zur Berechnung dieser Heuristik vorgestellt und analysiert. Anschließend wird ein Greedy-Algorithmus formuliert, der eine heuristische Lösung des Cograph Editing Problems in O(|V|^4) Zeit und unter Nutzung von O(|V|^2) Speicher berechnet. Eine Implementierung dieses Greedy-Algorithmus wird auf zufällig generierten Testdaten untersucht. Parallel dazu wird auch eine Implementierung für die korrekte Lösung des Cograph Editing Problems getestet. Dabei werden die Qualität der Ergebnisse und die Laufzeit gemessen und verglichen. Hier zeigte sich, dass der Greedy-Algorithmus bei der Rekonstruktion von durch Rauschen verzerrter Cographen vergleichbar gute Ergebnisse erzielen konnte.

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