Riboflavin transport is mediated, in part, by riboflavin transporter proteins 2 and 3, encoded by SLC52A2 and SLC52A3, respectively. Biallelic mutations in SLC52A2 and SLC52A3 impair riboflavin transporter protein function and riboflavin transport, causing disruptions to mitochondrial metabolism which result in sensory and motor neurodegeneration and give rise to riboflavin transporter deficiency (RTD) in humans. RTD is a rare neurodegenerative disease characterised by respiratory compromise, muscle and limb weakness, and vision and hearing impairments. RTD patients are treated with high-dose riboflavin supplementation which is effective in over 70% of cases but can be ineffective due to rapid excretion of riboflavin when its plasma concentration exceeds 0.5 μM. To address the need for alternative or supplemental RTD treatment, this study generated morpholino-mediated knockdown and CRISPR/Cas9 models of RTD in zebrafish. An RTD-like phenotype is observed in these RTD models including hearing loss, decreased motor axon length, and impaired locomotor activity. The slc52a3 morphant phenotype was found to be specific via coinjection of slc52a3 morpholino/human SLC52A3 mRNA, which achieved effective rescue of the morphant phenotype, as well as slc52a3 morpholino/p53 morpholino coinjection, which maintains the slc52a3 morphant phenotype. In line with clinical findings, riboflavin supplementation resulted in some improvement of the morphant phenotype. Probenecid was selected as a candidate drug due to its inhibitory effect on OAT-3, which mediates riboflavin excretion. However, supplementing riboflavin treatment with probenecid provided no additional benefit to the slc52a3 knockdown model. Further development of CRISPR/Cas9-knockout lines of slc52a2 and slc52a3, as well continued therapeutic screening of riboflavin and probenecid and consideration of alternative therapeutics will provide more opportunities to uncover novel therapeutic strategies to improve RTD treatment.

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.

Wnt signaling plays important role in many aspects of embryogenesis such as cell proliferation, cell fate specification, cell polarity and organogenesis(Clevers 2006, van Amerongen and Nusse 2009). Wnt ligands have been shown to activate several intra-cellular signaling cascades, including the canonical or Wnt/-catenin dependent pathway and the non-canonical or -catenin independent pathway. Dishevelled (Dvl) occupies a key position at crossroads of all branches of Wnt signaling cascade. To understand, how Dishevelled (Dvl) may channel signaling into the downstream branches, we sought to identify novel effectors for Dishevelled (Dvl) using a yeast-two hybrid screen. In this study, we used the PDZ domain of Dishevelled (Dvl) as a bait and from this screen, we identified a new binding protein of Dishevelled (Dvl)-termed as Custos. To characterize the functional role of Custos in Wnt signaling pathway, we used mammalian cell culture and zebrafish as a model vertebrate organism. We confirmed the interaction between Custos and Dvl using co-immunoprecipation and GST pull-down. Custos also interacted with -catenin in vivo and this interaction was positively regulated by Wnt stimulation. Immunofluorescence experiments in mammalian cells showed that Custos co-localizes with the nuclear envelope marker, lamin and inhibits translocation of -catenin to the nucleus. In zebrafish embryos, Custos is a maternal gene and expressed throughout development. Spatial in situ hybridization studies showed that Custos was expressed in the dorsal region of the embryo at early stages and in the nervous system in zebrafish at 24hpf. To delineate the biological role of Custos during embryogenesis, we conducted a gain of function and loss of function studies. Overexpression of exogenous Custos and morpholino knockdown of Custos revealed that Custos is critical for embryonic patterning. Knockout of Custos in zebrafish revealed that Custos delays embryonic development and exhibits defects in pigmentation suggesting a plausible role in neural crest development. Taken together, our studies demonstrate that Custos is a novel component of canonical Wnt signaling and required for -catenin translocation into the nucleus and important for embryonic patterning. / Biology

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23521号 / 農博第2468号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5352(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 准教授 坂本 正弘 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The pig has similar features to the human in aspects such as physiology, immunology, and organ size. Because of these similarities, genetically modified pigs have been generated for xenotransplantation. Also, when using the pig as a model for human diseases (e.g. cystic fibrosis transmembrane conductance regulator), the pig exhibited similar symptoms to those that human patients present. The main goal of this work was to examine the efficacy of direct injection of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) in pigs and to overcome shortcomings that resulted after direct injection into the cytoplasm of developing zygotes. By using direct injection of CRISPR/Cas9 into developing zygotes, we successfully generated fetuses and piglets containing 9 different mutations. The total number of aborted fetuses was 20 and of live piglets was 55. Moreover, one issue that was encountered during the production of mutated pigs was that insertion or deletion (indel) mutations did not always introduce a premature stop codon because it did not interfere with the codon read. As a result of these triplet indel(s) mutations, a hypomorphic phenotype was presented; consequently, the mutated gene was partially functional. To prevent this hypomorphic phenotype, we introduced two sgRNAs to generate an intended deletion that would remove a DNA fragment on the genome by causing two double-strand breaks (DSB) during non-homologous end joining (NHEJ). The injection of two sgRNAs successfully generated the intended deletion on the targeted genes in embryos and live piglets. Results after using intended deletions, in IL2RG mutation pigs, did not show hypomorphic phenotypes even when a premature stop codon was not present. After using the intended deletion approach, function of the targeted genes was completely disrupted regardless of the presence or absence of a premature stop codon. Our next aim was to introduce (i.e. knock-in) a portion of exogenous (donor) DNA sequence into a specific locus by utilizing the homology direct repair (HDR) pathway. Because of the cytotoxicity of the linear form of the donor DNA, the concentration of the injected donor DNA was adjusted. After concentration optimization, four different donor DNA fragments targeting four different genes were injected into zygotes. Efficiency of knock-in was an average of 35%. Another donor DNA was used in this study which is IL2RG-IA donor DNA carried 3kb of exogenous cassette. It showed 15.6% of knock-in efficiency. IL2RG-IA Donor DNA injected embryos were transferred into surrogates, and a total of 7 pigs were born from one surrogate, but none of the 7 were positive for the knock-in. Future experiments need to be developed to optimize this approach. Overall, the direct injection of CRISPR/Cas9 is advantageous in cost, time, and efficiency for large animal production and for biomedical research. However, there are still unsolved challenges (off-targeting effects, low efficiency of knock-in, and monoallelic target mutation) that need to be elucidated for future application in humans and other species. / Doctor of Philosophy / The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system is commonly used to make genetically modified pigs. The CRISPR/Cas9 system can break the DNA on a desired gene region. During the DNA repair process, random DNA base pairs can be inserted or deleted on the broken regions, thus generating a mutation on the desired gene. Scientists have adopted new methods to disrupt genes in many species. One of these new methods is the direct injection of CRISPR/Cas9 into a fertilized oocyte. In our first project, we used direct injection of the CRISPR/Cas9 system into the fertilized one-cell embryo. A total of 55 live pigs and aborted 20 fetuses with specifically disrupted genes were produced for biomedical research model. During these studies, one critical drawback of the direct injection method was encountered. Partial function of the gene was possible. To prevent this problem, two DNA broken regions were generated by the CRISPR/Cas9 system to remove the middle part the DNA by two DNA breaking. This method successfully removed the middle portion of the DNA targeted region in the pig embryos. Embryos injected with the CRISPR/Cas9 system to cut the two specific DNA regions were transplanted into surrogate pigs, and a total of 15 piglets were produced. All 15 pigs confirmed that a specific part of the gene had been removed by two DNA breakage. Also, no function of the desired gene was found in the 15 pigs. The objective of the last experiment was to introduce a specific exogenous DNA sequences into specific region of DNA using the CRISPR/Cas9 system. For this study, four different exogenous DNA fragments were synthesized for four different genes. When injected, one exogenous DNA along with the CRISPR/Cas9 system, the average integration efficiency of the four exogenous DNA fragments was 35% in the embryo. Another exogenous DNA, which was longer than other four DNA fragments showed 15.6% integration efficiency. The embryos injected with the long exogenous DNA fragment, along with the CRISPR/Cas9 system, were transferred into surrogate pigs. The result was that a total of 7 piglets were born, but the exogenous DNA sequence was not found in none of the seven piglets. In conclusion, the CRISPR/Cas9 system showed effective removal of the entire gene function of specific genes in the pig. However, for future application in the human and other species, some problems (un-wanted region mutation and low efficiency of exogenous DNA integration) continue to emerge and need to be addressed in future experiments.

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.

Les alphavirus sont un groupe de virus enveloppés à ARN simple brin positif retrouvés sur la totalité du globe et responsables de nombreuses maladies humaines et animales. Durant la dernière décennie, une réémergence du virus du chikungunya (CHIKV) a été observée causant de nombreuses épidémies sur tous les continents. Malgré les nombreuses études, les mécanismes moléculaires de réplication du CHIKV et les interactions hôte-virus restent peu caractérisées. L’objectif de mon travail était de mieux comprendre et caractériser l’entrée du virus du chikungunya et les facteurs de l’hôte impliqués dans la réplication chez les mammifères. Plusieurs approches distinctes ont été utilisées dans ce projet. Dans un premier temps, nous avons mis en avant une diminution de l’infection du CHIKV après un traitement avec du fer sous forme de citrate d’ammonium ferrique et nous avons étudié le rôle potentiel dans l’entrée virale de NRAMP2 et TFRC, deux protéines impliquées dans le transport cellulaire du fer et connus comme récepteurs d’entrée de plusieurs virus. D’autre part, nous nous sommes intéressés à deux autres protéines, CD46 et TM9SF2, identifiés à travers un criblage par ARNi réalisé en collaboration, dans le but de déterminer si elles sont utilisées comme facteurs d’entrée par le virus du chikungunya. Dans un dernier axe, nous avons mis en place et réaliser un criblage perte de fonction sur le génome entier en utilisant la technologie CRISPR/Cas9 afin d’identifier des facteurs de l’hôte importants pour l’entrée du CHIKV, sa réplication ou la mort viro-induite. Bien qu’il soit apparu que l’approche utilisée pour le criblage devrait être optimisée, nous avons pu identifier des candidats potentiellement nécessaires pour l’infection par le CHIKV. Ces candidats sont testés individuellement afin de confirmer leur implication dans la biologie du virus / Alphaviruses are a group of enveloped, positive-sense RNA viruses which are distributed almost worldwide and are responsible for a considerable number of human and animal diseases. Among these viruses, the Chikungunya virus (CHIKV) has recently re-emerged and caused several outbreaks on all continents in the past decade. Despite many studies, molecular mechanisms of chikungunya virus replication and virus-host interactions remain poorly understood. The aim of my project was to better understand and characterize the CHIKV entry and the host factors involved during replication steps in mammals. Several different approaches have been used in this work. As a first step, we have demonstrated a decrease of CHIKV infection after iron treatment in form of ferric ammonium citrate and we have studied the potential role in viral entry of NRAMP2 and TFRC, two proteins involved in iron transport and known receptors for other viruses. On the other hand, we have also focused on two proteins, CD46 and TM9SF2, identified through an RNAi screen in collaboration, in order to determine if they are required as entry factors for chikungunya virus. In a last axis, we have set up and carried out a genome-wide loss of function screen with the CRISPR/Cas9 technology in order to identify host factors important for chikungunya virus entry, replication or virus-induced cell death. Although it appears that screen conditions should be optimized, we have identified potential candidates required for CHIKV infection and we are currently testing them

O splicing do pré- RNAm é o processo pelo qual os introns são removidos e os exons são unidos para produzir um RNAm maduro. Esse processo é catalisado por um complexo denominado spliceossomo. RNF113A é uma proteína detectada em complexos pré- e pós-catalíticos. Neste trabalho analisamos a participação de RNF113A no spliceossomo de mamíferos e suas interações com outros componentes do spliceosomo. Nossos resultados mostraram que a super- expressão de RNF113A em células HEK293T aumentam o nível dos snRNAs U1, U2, U5 e U6 e de PRPF19. A imunoprecipitação de RNF113A revelou maior concentração de PRPF19, hBRR2 e SF3b2. Além disso, observamos maior quantidade dos snRNAs U5 e U6 associados à RNF113A. As frações de imunoprecipitação foram analisadas por espectrometria de massas e mostraram interação de RNF113A com proteínas que participam na transcrição, ubiquitinação, maturação do pré-RNAm e splicing. Estes resultados indicam que a associação de RNF113A ao spliceossomo ocorre a partir de suas interações com os snRNAs U5 e U6 e com proteínas relacionadas, como a hBRR2. / Splicing of pre-mRNA is the process by which introns are removed and exons are joined together to produce a mature mRNA competent for translation. It is catalyzed by the complex called spliceosome. RNF113A is a protein detected in pre- and post-catalytic complexes. In this work we analyzed RNF113A participation on spliceosome and its interactions with other spliceosome components. Our results showed that overexpression of RNF113A in HEK293T led to increased levels of U1, U2, U5, U6 snRNAs and PRPF19. RNF113A also associates to PRPF19, hBRR2 and SF3b2, as observed by immunoprecipitation followed by RT-qPCR and western blot. Consistently, U5 and U6 snRNAs were observed in association with RNF113A. Immunoprecipitation fractions were also analyzed by mass spectrometry and results showed interaction of RNF113A with proteins that participate in processes such as transcription, ubiquitination, pre-mRNA maturation and splicing. These results suggest RNF113A associates to spliceosome by interacting with U5 and U6 snRNAs and with related proteins, for example hBRR2.

With the increase of agricultural production, there is an improvement in the use of mineral fertilizers, which may cause different environmental problems, besides the soil salinization. A possible alternative for reducing the application of these products is the use of plant growth-promoting bacteria (PGPB), that can be used alone or in co-inoculation, resulting in an alternative environmentally and economically feasible. Better results can be obtained if the interaction among bacteria-bacteria and bacteria-plant be elucidated, and strategy developed to optimize these interactions. Thus, the plant growth-promoting Bacillus sp. RZ2MS9, previous described as a potential PGPB in maize and soybean, was GFP-tagged and monitored alone and co-inoculated with Azospirillum brasilense (Ab-v5::pWM1013) during maize colonization. The interaction of tagged strains in maize were monitored by fluorescent microscopy (FM) and quantitative PCR (qPCR), demonstrating an endophytic behavior of Bacillus sp. RZ2MS9. Although the non-detection of Ab-v5::pWM1013, the co-inoculation resulted in the best increase in root and shoot dried weight, root volume and in root diameter, showing that inoculation with more than one strain can be a good choice to development of bio-fertilizers. One important system to bacterial interaction is the quorum sensing (QS). The QS is an important cell-cell communication system that allows bacterial cells to recognize their own population and modulate their gene expression. This, system is also involved in the interspecific communication, including other bacterial species and plants. In the other hand, enzymes able to detect and degrade these molecules evolved, the called quorum quenching (QQ) system, that has been evolved in some bacteria as competitive advantage for niches colonization. The aiiA gene, was one of the first gene related with the QQ in Bacillus. The aiiA was found in Bacillus sp. RZ2MS9 genome. Through construction of a new QQ biosensor, Agrobacterium tumefaciens At11006, and validated by A. tumefaciens NTL4, the ability of RZ2MS9 to degrade QS molecules was confirmed. The knockout of aiiA gene was performed using the CRISPR-Cas9 system, confirming this gene function. By these results, the influence of QQ system of Bacillus sp. RZ2MS9 during maize colonization and RZ2MS9 - A. brasilense - maize can be better investigated, opens the possibility to better understand the role of QQ system in the interaction among PGPB and plants. / Concomitantemente ao aumento da produção agrícola, há o aumento do uso de fertilizantes minerais, que pode acarretar no desenvolvimento de diferentes problemas ambientais, além de causar a salinização dos solos. Uma possível alternativa para tentar reduzir a aplicação desses produtos é o uso de bactérias promotoras de crescimento de plantas (BPCPs), que podem ser usadas isoladamente ou em co-inoculação com outras bactérias, tornando-as uma alternativa ambientalmente e economicamente viável. Melhores resultados podem ser obtidos se a interação bactéria-bactéria e bactéria-planta for elucidada, permitindo que estratégias sejam desenvolvidas para otimizar essas interações. Em vista disso, a bactéria Bacillus sp. RZ2MS9, previamente descrita como uma potencial BPCP em milho e soja, foi marcada com GFP e monitorada durante a colonização de milho inoculada sozinha, bem como em co-inoculação com Azospirillum brasilense (Ab-v5::pWM1013). A interação dessas linhagens marcadas em milho, foi monitorada por microscopia de fluorescência (FM) e PCR quantitativo (qPCR), revelando um comportamento endofítico de Bacillus sp. RZ2MS9. Em plantas co-inoculadas, apesar da linhagem Ab-v5::pWM1013 não ter sido detectada por qPCR, a co-inoculação resultou no aumento do peso seco das raízes e da parte aérea, no volume e no diâmetro do sistema radicular, demonstrando que a inoculação com mais de uma linhagem bacteriana pode ser uma boa alternativa para o desenvolvimento de bio-fertilizantes. O quorum sensing (QS) é um importante sistema de comunicação célula-célula que permite que as bactérias reconheçam sua própria população e modulem sua expressão gênica. Este sistema também está envolvido na comunicação interespecífica, incluindo outras espécies bacterianas e plantas. Co-evolutivamente, enzimas capazes de detectar e degradar essas moléculas evoluíram, dando origem ao chamado quorum quenching (QQ), sistema que evoluiu em algumas bactérias como uma vantagem competitiva para a colonização de nichos. O gene aiiA, foi um dos primeiros genes relacionados ao sistema QQ descrito no gênero Bacillus, gene este que foi anotado no genoma de RZ2MS9. Através da construção de uma nova linhagem biossensora de QQ, Agrobacterium tumefaciens At11006, e validada através da linhagem A. tumefaciens NTL4, a capacidade de RZ2MS9 de degradar moléculas de QS foi confirmada. O knockout do gene aiiA foi realizado utilizando o sistema CRISPR-Cas9, confirmando a função desse gene. Através dos resultados obtidos neste trabalho, a influência do sistema QQ de Bacillus sp. RZ2MS9 durante a colonização do milho, bem como a interação RZ2MS9 - A. brasilense - milho pode ser melhor investigada, abrindo a possibilidade de uma melhor compreensão do papel do sistema QQ na interação entre bactérias promotoras de crescimento e plantas.

Le positionnement des nucléosomes le long des génomes eucaryotes est crucial étant donné qu’il affecte l’accessibilité de l’ADN à des protéines impliquées dans la transcription, la réplication, ou encore la réparation de l’ADN. Si il est aujourd’hui admis que les remodeleurs de chromatine ainsi que les préférences des nucléosomes pour certains motifs d’ADN constituent les deux principaux déterminants du positionnement des nucléosomes in vivo, leur importance relative fait encore l’objet de controverses. Dans le cadre de cette problématique nous avons développé une stratégie d’assemblage de répétitions de la séquence 601 positionnante de nucléosome directement dans le génome de Saccharomyces cerevisiae. Cette technique assistée par la technologie CRISPR/Cas9 et des oligonucléotides chevauchants s’est révélée très efficace et a permis d’assembler des répétitions sur une étendue d’environ 15 kilobases. Nous avons ainsi pu isoler trois souches se caractérisant par trois longueurs d’ADN de liaison de respectivement 20, 50 et 90 paires de bases séparant deux 601 consécutifs tout le long des répétitions. Ces longueurs d’ADN de liaison ont été choisies du fait de leur compatibilité avec les modèles de la fibre de 30 nm étudiés in vitro et parce qu’elles sont fréquemment observées chez les eucaryotes. Nous avons ensuite regardé si ces répétitions de la séquence 601 suffisent à dicter la succession des nucléosomes de S. cerevisiae selon le pas de chromatine attendu. Pour cela, nous avons eu recours à une approche de MNase-seq afin d’analyser les positions des dyades des nucléosomes dans les répétitions. Les résultats de ces analyses révèlent de façon intéressante l’incapacité de la séquence 601 à positionner le nucléosome dans ce contexte cellulaire et cela malgré l’étendue de la région de 601 répétés constituée. Nous avons également analysé le positionnement des nucléosomes chez ces trois mêmes souches suite à l’inactivation de Chd1, l’un des deux principaux architectes du paysage nucléosomal chez la levure, afin de s’affranchir de son potentiel effet sur le positionnement des nucléosomes dans la région 601. Nos résultats montrent que l’absence de Chd1 ne permet pas de rétablir un positionnement des nucléosomes sur les monomères de 601, suggérant que le 601 n’est pas positionnant in vivo ou que la région répétée est sous l’influence d’autres facteurs de remodelage. D’un point de vue méthodologique, notre technique de construction de répétitions in vivo permet d’envisager des approches simplifiées de biologie synthétique pour la construction de librairies de répétitions dans le génome de S. cerevisiae. / Nucleosome positioning along eukaryotic genomes is crucial as it influences DNA accessibility for DNA binding proteins involved in DNA replication, transcription and repair. It is now accepted that both nucleosome preferences for some DNA sequences and remodeling factors play an important role in nucleosome positioning in vivo. However their relative importance remains a matter of debate. To investigate the role played by DNA sequence in nucleosome positioning in a cellular context we developped a strategy to assemble tandem DNA repeats of a nucleosome positioning sequence directly into Saccharomyces cerevisiae’s genome. This method is assisted by CRISPR/Cas9 and overlapping oligonucleotides and it turned out to be very efficient as it allowed to synthetize about 15 kilobases of tandem DNA repeats inside a yeast chromosome. Using this apporoach we obtained three yeast strains differing by the DNA linker length separating two consecutive monomeres of the 601 nucleosome positioning sequence. We chose three lengths of linker (20, 50 and 90 pb) for two reasons. First, they are compatible with the formation of a 30 nm chromatin fiber in vitro, and second, nucleosome repeat length of 167, 197 and 237 pb are found in eukaryotic genomes. We then verified if nucleosomes are effectively positioned according to the theoretic DNA linker lengths we designed in the “601” repeated region. To that goal we performed MNase-seq analysis to deduce nucleosomes dyads positions in the repeats. Interestingly our results show that the 601 sequence is not able to dictate strong nucleosomes positioning differing from the natural nucleosome repeat length of about 165 pb along the repeats in an in vivo context. We further investigated positions of dyads in the same three strains after inactivating the gene coding for the chromatin remodeler Chd1, which could potentially be responsible of the nucleosomes organization in the repeated area. Our results show no effect of Chd1, indicating that the “601” sequence has no positionning effect in vivo or that other trans-acting factors are implicated in nucleosome positioning in the engineered repeats. Finally, this work provides a new fast and simple approach for synthetic DNA repeats construction inside the yeast genome and could easily be applied for other synthetic chromatin engineering approaches.