Genome editing mediated by engineered nucleases, including Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) systems, holds great potential in a broad range of applications, including biomedical studies and disease treatment. In addition to creating cell lines and disease models, this technology allows generation of well-defined, genetically modified cells and organisms with novel characteristics that can be used to cure diseases, study gene functions, and facilitate drug development. However, achieving both high efficiency and high specificity remains a major challenge in nuclease-based genome editing. The objectives of this thesis were to optimize the design of TALENs to achieve high on-target cleavage activity, and analyze the off-target effect of CRISPR/Cas to help achieve high specificity. Based on experimental evaluation of >200 TALENs, we compared three different TALEN architectures, proposed new TALEN design rules, and developed a Scoring Algorithm for Predicting TALEN Activity (SAPTA) to identify optimal target sites with high activity. We also performed a systematic study to demonstrate the off-target cleavage by CRISPR/Cas9 when DNA sequences contain insertions or deletions compared to the RNA guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis, and suggest specific guidelines for reducing potential off-target cleavage of CRISPR/Cas9 systems. The studies performed in this thesis work provide important insight and powerful tools for the optimization of engineered nucleases in genome editing, thus making a significant contribution to biomedical engineering and medical applications.

A inserção de DNA exógeno no genoma hospedeiro é conseguida principalmente através da utilização de vias de reparo como a junção de pontas não homólogas, que possui caráter aleatório, e a recombinação homóloga, que possibilita o gene targeting. Algumas ferramentas como as TAL Effector Nucleases (TALENs) e o RNA interferência (RNAi) podem ser utilizadas para aumentar a taxa de integração específica e assim melhorar a eficiência e o direcionamento da edição gênica. Nesse trabalho utilizamos o silenciamento gênico mediano por short interference RNA (siRNA) para inibição temporária dos genes ATF7IP uma metiltrasferase, EP300 uma acetiltransferase e KU70 (NHEJ) e um par de TALENs complementares a uma região do gene da distrofina canina. Células Caninas MDCK I foram transfectadas por lipofectamina 2000 (Invitrogen) com 320pmol de siRNAs para ATF7IP e Ep300; e 64 pmol do SiRNA para KU70 em diferentes grupos, 40 horas depois as células foram transfectadas com 15 μg vetor molde derivado do pEGFP-N1 (Clonatech) e com 10 μg dos RNAm das TALENs. A seleção se deu em meio DMEM high com 600μg/ mL de G418 (Lonza) por 14-16 dias. As colônias coletadas através de biópsias foram analisadas por Polimerase Chain Reaction e sequenciamento gênico. Três pares de primers foram utilizados; um controle endógeno (GAPDH), um controle interno do inserto (Neo qPCR) e um para confirmação da recombinação homóloga (DMD3). Os grupos apresentaram grande variação na taxa de mortalidade celular e consequentemente no número de colônias: Com o grupo ATF7IP+Vetor (648c) apresentando maior número de colônias e o grupo EP300+Ku70+Vetor+TALENs o menor (1c). A maior taxa de recombinação ocorreu nos grupos no grupo ATF7IP +Ku70+Vetor+TALENs com 40% das células positivas para neomicina apresentado o evento gene targeting, um aumento considerável na taxa de recombinação quando comparada a porcentagem de 3,1% do controle transfectado somente com o vetor molde. Mostrando que o uso conjunto das TALENs com siRNAs foi um sucesso para o aumento de eventos de edição gênica direcionada. / The insertion of exogenous DNA into a host genome is achieved primarily through the use of DNA repair pathways such as Non-Homologous End Joining (NHEJ) and the Homologous Recombination (HR). The integration by NHEJ has a random feature and is much more common than HR insertions, which are more likely to produce gene targeting events . TAL effector nucleases (TALENs) and RNA interference (RNAi) can be used to increase the rate of specific integration and thus improving the efficiency of gene editing. In this work, we used short interference RNA (siRNA)-mediated gene silencing for transient inhibition of genes ATF7IP (implicated in histone methylation), EP300 (acetyltransferase) and Ku70 (essential to NHEJ) and a pair of TALENs RNAm complementary to canine muscle dystrophin (DMD) gene. MDCK I Canine Cells were transfected by lipofectamine 2000 (Invitrogen) with 320 pmol of siRNAs for ATF7IP and EP300; and 64 pmol of siRNA for Ku70 in different groups. After 40 hours cells were transfected with 15 μg of a vector derived from pEGFP- N1 (Clontech) containing two regions homologous to the canine DMD gene (left arm length: 873 bp and right arm length: 1370 bp) and 10 μg of TALEN mRNA. The cell selection was achieved with DMEM high glucose with 600μg/ml G418 for 14-16 days. The colonies collected through biopsies were analyzed by polymerase chain reaction and gene sequencing. Three pairs of primers were used; an endogenous control (GAPDH) , an internal control of the insert (Neo qPCR) and a primer set to confirm the occurrence of homologous recombination events (DMD3). .Groups showed great variation in cell death rate and consequently in the number of colonies: ATF7IP+Vector had highest number of colonies (648c) and the group EP300+Ku70+Vetor+TALENs the lowest one (1c) The highest rate of homologous recombination was in ATF7IP +Ku70+Vetor+TALENs group that had 40% of the neomycin positives cells confirmed as gene targeting events, a considerable increase in the recombination rate compared to the 3.1% in the control group transfected only with the template vector. That shows that the combined use of siRNAs and TALENs was a success for increasing directed gene editing events.

A inserção de DNA exógeno no genoma hospedeiro é conseguida principalmente através da utilização de vias de reparo como a junção de pontas não homólogas, que possui caráter aleatório, e a recombinação homóloga, que possibilita o gene targeting. Algumas ferramentas como as TAL Effector Nucleases (TALENs) e o RNA interferência (RNAi) podem ser utilizadas para aumentar a taxa de integração específica e assim melhorar a eficiência e o direcionamento da edição gênica. Nesse trabalho utilizamos o silenciamento gênico mediano por short interference RNA (siRNA) para inibição temporária dos genes ATF7IP uma metiltrasferase, EP300 uma acetiltransferase e KU70 (NHEJ) e um par de TALENs complementares a uma região do gene da distrofina canina. Células Caninas MDCK I foram transfectadas por lipofectamina 2000 (Invitrogen) com 320pmol de siRNAs para ATF7IP e Ep300; e 64 pmol do SiRNA para KU70 em diferentes grupos, 40 horas depois as células foram transfectadas com 15 μg vetor molde derivado do pEGFP-N1 (Clonatech) e com 10 μg dos RNAm das TALENs. A seleção se deu em meio DMEM high com 600μg/ mL de G418 (Lonza) por 14-16 dias. As colônias coletadas através de biópsias foram analisadas por Polimerase Chain Reaction e sequenciamento gênico. Três pares de primers foram utilizados; um controle endógeno (GAPDH), um controle interno do inserto (Neo qPCR) e um para confirmação da recombinação homóloga (DMD3). Os grupos apresentaram grande variação na taxa de mortalidade celular e consequentemente no número de colônias: Com o grupo ATF7IP+Vetor (648c) apresentando maior número de colônias e o grupo EP300+Ku70+Vetor+TALENs o menor (1c). A maior taxa de recombinação ocorreu nos grupos no grupo ATF7IP +Ku70+Vetor+TALENs com 40% das células positivas para neomicina apresentado o evento gene targeting, um aumento considerável na taxa de recombinação quando comparada a porcentagem de 3,1% do controle transfectado somente com o vetor molde. Mostrando que o uso conjunto das TALENs com siRNAs foi um sucesso para o aumento de eventos de edição gênica direcionada. / The insertion of exogenous DNA into a host genome is achieved primarily through the use of DNA repair pathways such as Non-Homologous End Joining (NHEJ) and the Homologous Recombination (HR). The integration by NHEJ has a random feature and is much more common than HR insertions, which are more likely to produce gene targeting events . TAL effector nucleases (TALENs) and RNA interference (RNAi) can be used to increase the rate of specific integration and thus improving the efficiency of gene editing. In this work, we used short interference RNA (siRNA)-mediated gene silencing for transient inhibition of genes ATF7IP (implicated in histone methylation), EP300 (acetyltransferase) and Ku70 (essential to NHEJ) and a pair of TALENs RNAm complementary to canine muscle dystrophin (DMD) gene. MDCK I Canine Cells were transfected by lipofectamine 2000 (Invitrogen) with 320 pmol of siRNAs for ATF7IP and EP300; and 64 pmol of siRNA for Ku70 in different groups. After 40 hours cells were transfected with 15 μg of a vector derived from pEGFP- N1 (Clontech) containing two regions homologous to the canine DMD gene (left arm length: 873 bp and right arm length: 1370 bp) and 10 μg of TALEN mRNA. The cell selection was achieved with DMEM high glucose with 600μg/ml G418 for 14-16 days. The colonies collected through biopsies were analyzed by polymerase chain reaction and gene sequencing. Three pairs of primers were used; an endogenous control (GAPDH) , an internal control of the insert (Neo qPCR) and a primer set to confirm the occurrence of homologous recombination events (DMD3). .Groups showed great variation in cell death rate and consequently in the number of colonies: ATF7IP+Vector had highest number of colonies (648c) and the group EP300+Ku70+Vetor+TALENs the lowest one (1c) The highest rate of homologous recombination was in ATF7IP +Ku70+Vetor+TALENs group that had 40% of the neomycin positives cells confirmed as gene targeting events, a considerable increase in the recombination rate compared to the 3.1% in the control group transfected only with the template vector. That shows that the combined use of siRNAs and TALENs was a success for increasing directed gene editing events.

Integration and expression of exogenous genetic material – in particular, transgenes – into the genomes of model organisms, cell lines or patients is widely used for the creation of genetically modified experimental systems and gene therapy. However, loss of transgene expression due to silencing is still a major hurdle which remains to be overcome. Judicious selection of integration loci can help alleviate the risk of silencing; in recent years the ability to efficiently and specifically target transgene integration has been improved by the advent of site-specific nucleases (SSNs). SSNs can be used to generate double strand breaks (DSBs) in a targeted manner, which increases the efficiency of homologous recombination (HR) mediated transgene integration into predetermined loci. In this work I investigate four human genomic loci for their potential to act as transgene integration sites which will support robust long term expression: the adeno-associated virus (AAV) integration site 1 (AAVS1); the human homologue of the mouse Rosa26 locus (hROSA26); the inosine monophosphate dehydrogenase 2 (IMPDH2) gene and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene. I also investigate the potential of creating a novel drug-selectable transgene integration system at the IMPDH2 locus to allow for rapid and specific selection of correctly inserted transgenes. In addition to their ability to drive targeted transgene integration, SSNs can be harnessed to specifically disrupt gene function through indel formation following erroneous repair of the induced DSB. Using this strategy, I aimed to answer some important biological questions about eukaryotic translation elongation factor 1 alpha (eEF1A); eEF1A is responsible for providing aminoacylated tRNAs to the ribosome during the elongation phase of protein synthesis. Humans and other vertebrates express two isoforms, eEF1A1 and eEF1A2 (encoded by EEF1A1 and EEF1A2 respectively). During development eEF1A1 is replaced by eEF1A2 in some tissues. The reasons for this remain elusive, but one explanation may lie in the moonlighting functions of eEF1A1, which may not be shared by eEF1A2. Additionally, eEF1A2 can act as an oncogene, while there is no evidence that eEF1A1 is overexpressed in tumours. To begin to untangle these issues I targeted EEF1A1 using SSNs with the aim of making a cell line expressing only the eEF1A2 isoform. This work suggests that eEF1A1 may be essential even in the presence of eEF1A2, though further studies will be required to confirm this.

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

Studiens syfte är att visa på hur simuleringsbaserade aktiviteter kan användas för att lära ut stora talens lag till elever på mellanstadiet. Studiens kan förhoppningsvis vidare kunna bidra till en ökad kunskap om hur elever kan lära sig om sannolikhet. För att uppnå syftet användes två olika frågeställningar. Frågeställningarna var följande: Hur kan elever på mellanstadiet utveckla sina kunskaper om de stora talens lag med hjälp av simuleringar? Vilka kritiska aspekter om sannolikhet är möjliga för eleverna att urskilja med hjälp av simuleringsaktiviteter? Vilka övriga aspekter av matematikundervisningen synliggörs under studiens gång?  Studien utgår ifrån variationsteorins syn på lärande och därav valdes metoden Learning study till studien. Variationsteorin belyser variationens betydelse för att möjliggöra ett lärande. Learning study syftar till att nå ett lärande vilket är i linje med det den här studien vill uppnå.  Resultatet i studien visar både att det både finns för- och nackdelar med arbete genom simulering. En fördel var att växande stickprov visade sig vara ett bra sätt för elever att urskilja olika kritiska aspekter. En nackdel var att vissa kritiska aspekter var svåra för eleverna att urskilja. Exempelvis var den kritiska aspekten om begreppen tur och skicklighet fortsatt en svårighet för många elever även efter studien.

L'application des techniques de séquençage à haut débit dans les dernières années a conduit à l'obtention de la séquence de génomes complets de plusieurs organismes. Le développement de nouveaux outils de génétique inverse était donc souhaitable afin de faire un usage optimal des données accumulées. Les nucléases hautement spécifiques représentent un outil unique pour induire des modifications ciblées du génome in vivo. L'induction d'une cassure double brin dans l'ADN est réparée soit par la voie de jonction d’extrémités nonhomologues soit par la voie très fidèle de la recombinaison homologue. Le ciblage d'un locus précis avec une nucléase spécifique stimule fortement la réparation de l'ADN, qui peut être utilisé pour induire des modifications ciblées dans le génome. Dans ma thèse, je vise à fournir une preuve de prinicipe pour l'utilisation des méganucléases et des transcription activator like effector nucléases (TALENs), deux classes communes de nucléases très spécifiques, comme nouveaux outils d'édition du génome chez le medaka, Oryzias latipes, et le poisson zèbre, Danio rerio. J'ai d’abord trouvé les conditions optimales d'utilisation de ces nucléases dans nos modèles de poissons. J'ai à cette occasion également développé une méthode très sensible et rapide pour la détection de modifications génomiques ciblées. J'ai ensuite induit des mutations au sein de trois gènes différents chez le poisson zèbre avec des TALENs. Les mutations dans le gène col6a1 ont conduit à la mise en évidence pour la première fois d’une technique de modification d’un site d’épissage d’un gène de poisson zèbre à l’aide d’une nucléase ciblée. Ce travail nous a permis d’établir une lignée de poisson avec une mutation dans le collagène VI alpha 1 qui est similaire à une mutation fréquemment trouvée chez les patients humains atteints de myopathie de Bethlem. De même, j’ai pu induire des mutations dans le gène nle1 du poisson zèbre qui vont permettre la mise en place de lignées de poissons mutants de ce gène. En outre, j'ai pu montrer qu’un nouveau type de nucléase, une TALEN Compact, était actif sur une cible chromosomique chez le poisson zèbre. En conclusion, les études que j'ai effectuées ont apporté la preuve de principe pour l'activité de TALENs et Compact TALENs ainsi que la première démonstration de modification de l'épissage à l’aide d’une TALEN chez le poisson zèbre et ont abouti à la mise en place d'une lignée de poisson dont le phénotype est proche d’un syndrome humain ouvrant la voie à la création de modèles pour d’autres mutations de ce type. Pour finir, je discute dans cette thèse des conditions permettant un usage le plus efficace des nucléases ciblées pour la génération de mutants et l’édition du génome. / The application of high throughput sequencing techniques in the recent years has led to obtaining the full genome sequences of many organisms. The development of novel tools for reverse genetics was thus desirable to make optimal use of the accumulated data. Site directed nucleases represent a unique platform to induce targeted genome modifications in vivo. Targeting a precise locus with a highly specific nuclease stimulates DNA repair, which can be harnessed for genome editing. Induction of a double strand break in DNA is repaired by either the error prone pathway of nonhomologous end joining or the high fidelity pathway of homologous recombination in the cell. Both mechanisms can be used to insert foreign DNA into the genome of the host. In my thesis, I aimed to provide proof of principle for the use of meganucleases and transcription activator like effector nucleases (TALENs), two common classes of site directed nucleases, as novel tools for genome editing in medaka, Oryzias latipes, and zebrafish, Danio rerio. During the first years of my thesis, I found the optimal conditions to use these nucleases in our fish models. I also developed a very sensitive and rapid method for detection of targeted genome modifications. I then induced mutations at three different endogenous loci in zebrafish with TALENs. The mutations in the col6a1 gene led to the first demonstration of splicing site modification in zebrafish using a TALE nuclease. This allowed the establishment of a fish line with a mutation in type VI collagen alpha 1 chain homologous to one mutation frequently found in human patients with Bethlem myopathy. Then I generated mutations in the nle1 gene which are heritable and from which establishment of mutant fish lines is in progress. In addition, by using the method for detection of targeted genome modifications I developed, I showed that a novel type of nuclease, a Compact TALEN, was active on a chromosomal target in zebrafish. In conclusion, the studies I performed provided proof of principle for the activity of TALENs and Compact TALENs as well as the first demonstration of TALEN-Mediated modification of splicing in zebrafish and resulted in the establishment of a fish line with mutated collagen VI. Induction of heritable mutations in the nle1 gene in zebrafish was also confirmed. Additionally, I proved that the choice of expression vector is crucial for the synthesis of active site directed nucleases for use in fish and established a novel efficient method for detection of targeted genomic mutations.

Ce travail de doctorat consiste en l’étude des propositions paradoxales dans la première partie de l’œuvre poétique de Jenaro Talens (1964-1991). A partir des deux notions de paradoxe et de poésie, nous avons organisé nos recherches autour des structures syntagmatiques et paradigmatiques de l’énonciation paradoxale, et de la spécificité de ce discours poétique que nous avons appelé axe poétique. Ainsi après avoir défini deux grandes catégories de paradoxes (logiques et étymologiques), nous avons analysé la structure et les constructions de la signifiance des propositions paradoxales pour révéler leur rôle et leur fonction dans l’œuvre étudiée : moteur même de l’écriture, le sentiment paradoxal, synonyme du désarroi, manifeste la limite de la structuration duelle du réel pour inviter à une organisation non-duelle du monde. / This work consists in the study of paradoxical clauses in the first part of Jenaro Talens’s poetic works (from 1964 to 1991). Starting from the two notions of paradox and poetry, we have organized our research around the syntagmatic and paradigmatic structures of the enunciation of paradoxes and the specificity of this poetic discourse, which we called poetic axis. So, after defining 2 large categories of paradoxes (logical and etymological), we have analysed the structure and the constructions of paradoxical propositions, in order to reach their role and function of the work under study : the feeling of paradox, as a synonym of disarray, which is the fuel of writing, shows the limit of the dual structure of the real, to lead to a non dual organisation of the world.

Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.

During meiosis eukaryotes produce four haploid gametes from a single diploid parental cell. In meiotic S-phase homologous chromosomes, which were inherited from maternal and paternal parents, are replicated. Homologous chromosomes then pair and undergo reciprocal crossover, which generates new mosaics of maternal and paternal sequences. Meiosis also involves two rounds of chromosome segregation, meaning that only one copy of each chromosome is finally packaged into the resulting haploid gametes. In this work I sought to genetically engineer two elements of meiosis, in order to generate tools which may be useful for plant breeding. The first project sought to generate a second division restitution (SDR) population, where the second meiotic division is skipped. This is created by crossing an SDR mutant, omission of second division1, which produces diploid pollen due to a defective meiosis-II, to a haploid inducer line, whose chromosomes are lost from the zygote post-fertilisation. This was intended to give rise to diploid plants possessing chromosomes from just the SDR parent. Importantly, the SDR parent used was heterozygous, meaning that SDR progeny should show mostly homozygous chromosomes, but with regions of residual heterozygosity, determined by crossover locations. This project succeeded in creating a small number of plants with the predicted SDR genotype, although a range of aberrant genotypes were also observed. I present several hypotheses that could account for the observed progeny genotypes. In a second project I attempted to direct meiotic recombination using DNA double strand breaks targeted to specific sites. This project used a spo11-1 mutant, which is unable to produce the endogenous meiotic DNA DSBs that normally mature into crossovers. Instead, TALFokI nucleases (TALENs) were expressed from meiotic promoters in order to generate exogenous DSBs at sites determined by the DNA binding specificity of the TAL repeat domains. The project succeeded in transforming TALENs into spo11-1 mutants and confirming their expression. However, this was not sufficient to recover the spo11-1 mutant infertility or direct crossovers. Potential reasons for this non-complementation are discussed, as well as their implications for control of meiotic recombination in plant genomes.