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

CRISPR/Cas has been developed for targeted mutagenesis in diverse species, including plants. However, precise genome editing via homology-directed repair (HDR) is inefficient in plants, limiting our ability to make large deletions or insertions in the plant genomes. Prime editing increases the control over the desired editing and allows the precise introduction of all types of mutations, including insertion, deletions, and all possible base conversions, albeit at low efficiencies. Here, we designed a dual prime editing system to generate large deletions and precise insertions of sequences by repairing template complementarity. We coupled dual pegRNA with Cas9 nickase (nCas9) to generate deletions and insertions. In another modality, we used dual pegRNA with wild-type Cas9 to generate double-stranded breaks to improve the editing at the targeted sites. We tested dual pegRNAs to delete the last exon in OsCCD7, delete the microRNA targeted sequence in OsIPA, and insert the T7 promoter in the 3'UTR of OsALS. Our results showed a high frequency of targeted insertion of the T7 promoter sequence in the 3'UTR of OsALS with wtCas9 and nCas9. Sanger sequencing analysis showed partial deletions at the targeted locus. Further improvements in the designs of pegRNAs will increase the precise genome insertions and deletions in plants.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22503号 / 農博第2407号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5283(農学部図書室) / 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 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

More than 300 human conditions, ranging from cancer predisposition to developmental and neurological mendelian disorders, are caused by haploinsufficiency (HI), a genetic condition by which mutational inactivation of a single allele leads to reduced protein levels and is enough to produce the disease phenotype. Therefore, translational enhancement of the spare allele could exert a therapeutic effect. Here we propose a novel approach for the potential rescue of haploinsufficiency disease loci based on the insertion of specific single nucleotide changes in the Kozak sequence. Since this sequence controls translation by regulating start codon recognition, we aimed at identifying and introducing specific nucleotide variations to enhance translation and rescue haploinsufficiency. To do so, we used CRISPR-Cas base editors, able to generate single nucleotide changes in genomic DNA without the need of a donor DNA and without creating double-strand breaks. We performed a high-throughput screening to evaluate the strength of the Kozak sequences of 231 haploinsufficient genes. We compared the translational efficiency of each wild-type sequence to that of several variants using FACS-seq, which combines fluorescence-activated cell sorting and high-throughput DNA sequencing. We thus selected 5 candidate genes (PPARGC1B, FKBP6, GALR1, NRXN1, and NCF1) and several nucleotide variations able to up-regulate translation. Finally, we used CRISPR-Cas base editors to reproduce the most efficient variants of NCF1 in a cell model relevant for the associated haploinsufficient disease and verified the increase of protein levels. This study proposes a novel therapeutic strategy to rescue haploinsufficiency and sheds new insights into the regulatory mechanisms underlying the translational process. On a broader level, the possibility of modulating gene expression by acting exclusively on translation expands the CRISPR-Cas genome editing applications.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24912号 / 農博第2575号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 准教授 小野 肇 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D.

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function. / Doctor of Philosophy / Within the mitochondria, “the powerhouse of the cell,” exists a series of five enzyme complexes that produce 90% of the energy for our cells need to function. The largest of these enzymes, complex I (CI), plays an important role in ensuring proper mitochondrial function. Injury to CI contributes to a number of diseases, but surprisingly few options exist to treat complex I. One of the most prevalent forms of CI dysfunction can be seen in ischemia/ reperfusion injury, a form of which is most commonly recognized as a heart attack. Surprisingly, the American Heart Association reports that in the next year over 700,000 people in the US will suffer from an ischemic event. With such a profound impact on the population, the need for new therapeutic developments is extremely high. Some current therapeutic approaches have been shown to be effective at treating cardiac dysfunction, but few address the dysfunction that occurs in the mitochondria. Here we test both a method for modeling these ischemia/reperfusion-based injuries and a potential therapeutic for treating these injuries within the context of CI dysfunction. We further evaluate CI dysfunction by using both established genetic engineering approaches as well as a completely new method to model CI disease. Through the use of two cutting edge genetic engineering approaches, we have been able to directly edit components of the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided a first-in-class method for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.

Im experimentellen Ansatz sollte mithilfe der CRISPR-Cas9-Methode eine gerichtete ALK1-Rezeptor-Eliminierung in myoblastischen C2C12-Zellen durchgeführt werden. Nach erfolgreicher Klonierung der jeweiligen, für den Typ-I-Rezeptor ALK1-kodierenden, gRNA-Sequenzen in die Puro- und GFP-CRISPR-Plasmide gelang der mittels Lipofektion durchgeführte Transfer der vier klonierten Plasmide in die C2C12-Zellen. Parallel aufgetaut wurden C2C12*ALK2- sowie ALK3-Knockout-Zelllinien, welche zuvor durch die Masterandin L. Wiesmann, ebenfalls mithilfe der CRISPR-Cas9-Methode, induzierte Knockouts der jeweiligen Rezeptoren ALK2 sowie ALK3 enthielten. Anschließend erfolgte die Puromycin-Selektion der mit den Puro-Klonen transfizierten C2C12*ALK1-3, ALK1-4-, ALK2- sowie ALK3-KO-Zellpopulationen. Die Zellen der C2C12*ALK1-3-KO-Population überlebten die Selektion trotz erneuter Durchführung der Transfektion sowie Selektion nicht. Somit erfolgte die Kultivierung der verbliebenen Zellen der C2C12*ALK1 4-, C2C12*ALK2- sowie C2C12*ALK3-KO-Population. Anschließend galt es zu untersuchen, wie responsiv die einzelne KO-Zelle für verschiedene Liganden ist. Im Rahmen der Durchführung differenter, zellbasierter Versuche wie der qPCR, des Western Blots und des ALP-Assays wirkten verschiedene BMPs auf die KO-Populationen ein. Somit konnten die BMP-induzierten, nachfolgenden Ereignisse wie die mRNA-Expression, die SMAD-Phosphorylierung sowie die Induktion der ALP-Expression innerhalb der KO-Populationen genauer betrachtet werden. Es ist allgemein bekannt, dass ALK1 sowohl bei der Angiogenese als auch bei der kardio-vaskulären Homöostase eine wichtige Rolle übernimmt. ALK1 ist vermutlich für die Gefäßneubildung in manchen Tumoren verantwortlich und auch die vaskuläre Erkrankung „Hereditäre hämorrhagische Teleangiektasie (HHT)“ steht im Zusammenhang mit einer Mutation des ALK1-Rezeptorgens. BMP9 beeinflusst als ALK1-bindender Ligand neben der Tumorentwicklung und der Angiogenese auch die osteogene Differenzierung mesenchymaler Stammzellen. Im Hinblick auf zukünftige Versuche sind daher weitere, noch aussagekräftigere Ergebnisse erstrebenswert, allerdings unter der Verwendung von ausschließlich homozygoten KO-Zelllinien. Weitere Erkenntnisse über die Rolle des ALK1-Rezeptors in BMP-vermittelter Signaltransduktion könnten für therapeutische Ansätze bei der Behandlung von vaskulären Erkrankungen und Tumorprogression sowie bei der Förderung der Knochenregeneration und -heilung hilfreich sein. / In the experimental approach, targeted ALK1 receptor elimination was to be performed in myoblastic C2C12 cells using the CRISPR-Cas9 method. After successful cloning of the respective gRNA sequences coding for the type I receptor ALK1 into the Puro and GFP-CRISPR plasmids, the four cloned plasmids were transferred into the C2C12 cells by lipofection. In parallel, C2C12*ALK2 and ALK3 knockout cell lines were thawed, which previously contained knockouts of the respective ALK2 and ALK3 receptors induced by the master student L. Wiesmann, also using the CRISPR-Cas9 method. Puromycin was then used to select the C2C12*ALK1-3, ALK1-4, ALK2 and ALK3-KO cell populations transfected with the Puro clones. The cells of the C2C12*ALK1-3-KO population did not survive the selection despite renewed transfection and selection. The remaining cells of the C2C12*ALK1-4, C2C12*ALK2 and C2C12*ALK3-KO populations were therefore cultivated. It was then necessary to investigate how responsive the individual KO cells are to different ligands. By performing different cell-based experiments such as qPCR, Western blot and ALP assay, different BMPs affected the KO populations. Thus, the BMP-induced downstream events such as mRNA expression, SMAD phosphorylation and induction of ALP expression within the KO populations could be analysed precisely.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25339号 / 農博第2605号 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms. In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing. Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.