Master of Science / Genetics Interdepartmental Program / Eduard D. Akhunov / The CRISPR/Cas9-based genome editing system holds a great promise to accelerate wheat improvement by helping us to understand the molecular basis of agronomic traits and providing means to modify genes controlling these traits. CRISPR/Cas9 is based on a synthetic guide-RNA (gRNA) that can guide Cas9 nuclease to specific targets in the genome and create double strand breaks (DSB). The DSB are repaired through the error-prone non-homologous end joining process causing insertions or deletions that may result in loss-of-function mutations. Here, we have developed an effective wheat genome editing pipeline. We used next-generation sequencing (NGS) data to estimate genome editing efficiency of many gRNAs using the wheat protoplast assay and choose the most efficient gRNAs for plant transformation. We successfully applied this pipeline to five wheat orthologs of the rice yield component genes that have been identified previously. We obtained edited plants for all these genes and validated the effect of the mutations in TaGW7 on wheat traits, which showed trends similar to those in rice. These results suggest that transferring discoveries made in rice to improve wheat is a feasible and effective strategy to accelerate breeding.

Human pluripotent stem cell (hPSC)-derived neuronal and glial material presents a relatively new opportunity to model human neurophysiology in both health, and disease. Validation of regionally-defined hPSC-derived neurones and glia cultures thus represents the founding blocks of technology that aims to complement existing models. Principally, the relevance of in vitro hPSC-derived material is determined by how representative it is of native material, yet at present the physiology of these cells remains underexplored. Here, electrophysiology and pharmacology are used to functionally assess hPSC-derived excitatory cortical neurones (hECNs), motorneurones (MNs) and oligodendrocyte-lineage cells in the context of regional-specific properties and maturation. These properties are then examined in material derived from hPSCs generated from patients with neurological disorders. This thesis examines of the properties of GABAARs and strychnine-sensitive glycine receptors (GlyRs) in hECNs by assessing their subunit composition, and compares these with studies which have made comparable investigations of rodent tissue where maturation is associated with a shift in GABAA and GlyR compositions. Using pharmacology and RNAseq analysis, GABAAR and GlyRs in hECNs were found to possess receptor populations typical of those reported in the immature cortex. hECNs generated from patients harbouring a mutation to the Disrupted-in-schizophrenia-gene 1 (DISC1), a candidate schizophrenia gene, were then examined. Imbalances in the excitation/inhibition balance are suspected in schizophrenia and, in this regard, the intrinsic excitability properties alongside expression and composition of major neurotransmitter receptors and intracellular chloride concentration were assessed. No obvious differences in excitability or functional expression of AMPARs, GABAARs or NMDARs were observed between case and control derived neurones. Receptor composition and intracellular chloride concentrations were found to be predominantly immature-like, however, AMPAR composition and intracellular chloride concentration were found to be like that of adult cortical neurones. These data are discussed in the context of modelling DISC1-associated pathologies. Thirdly, MNs from hPSCs generated from ALS patients harbouring mutations on the C9ORF72 gene were examined. The hypothesis that increased glutamate-mediated excitoxicity could, in part, be explained by increased expression of Ca2+- permeable AMPARs was examined. The estimated mean single-channel conductance of AMPARs was found to be high in MNs derived from ALS patients, reminiscent of Ca2+-permeable AMPARs and was reversed by gene-editing of the C9ORF72 mutation. Finally, oligodendrocytes generated from ALS patients harbouring TARDBP mutations were examined. Distinctive electrophysiological shifts in oligodendrocytes-lineage cell development are reported. A similar AMPAR phenotype of elevated Ca2+-permeable AMPAR expression was observed in oligodendrocytes derived from two patient hPSC lines and was rescued in an isogenic, gene-edited line, raising the intriguing possibility of convergence in pathophysiologies in the nature of the overlap between cell-type, AMPAR pathology and excitotoxicity in ALS disease progression mechanisms.

Primary liver cancer (PLC) is the second most common cause of cancer death worldwide, preceded only by lung cancer. Current models for PLC either fail to fully recapitulate tumour histology and architecture or are expensive, time consuming and do not allow for personalised drug testing. During the first part of my PhD, I have collaborated with Dr. Laura Broutier in order to established a new 3D in vitro model system for liver cancer. Based on the current knowledge on organoid cultures, we have managed to establish a system to grow primary human liver cancer cells long-term (Broutier et al., in press). Interestingly, the tumour-derived organoids (tumoroids) recapitulate the original tumour histology and genetic alterations and are also able to generate tumours in an in vivo xenograft mouse model after long-term expansion. Furthermore, we have shown that tumoroids can also be successfully used for drug testing, suggesting their use to devise new targeted therapy as well as personalised treatment strategies. Current models to investigate the role of genes in cancer rely mostly on animal studies, which can be very time consuming and cost demanding, especially if resulting in negative outcomes. To overcome this issue, I have set up a protocol for introducing mutations in healthy human liver organoids using the CRISPR-Cas9 technology. Interestingly, after mutating TP53, RNF43 and ZNRF3 either alone or in combination, human organoids undergo genetic alterations and phenotypic changes that partially resemble the ones observed in tumoroids. This data suggests that this system could be used as a screening platform to study gene function before using animal models. In the last part, I have further explored the role of RNF43 and ZNRF3 (R&Z) - two newly identified WNT pathway negative regulators mutated in many cancer types - in the liver using an in vivo mouse model. Interestingly, conditional deletion of R&Z specifically in adult mouse hepatocytes results metabolic changes that eventually lead to extensive liver damage. However, when the liver is challenged to regenerate in a chronic damage model, R&Z mutated livers fail to fully repair and show presence of multiple regenerative nodules. Later, livers develop either focal nodular hyperplasia and/or early hepatocellular carcinoma. These data suggest that R&Z have an important role in both liver metabolic homeostasis and liver regeneration and that their alteration can eventually lead to cancer formation.

abstract: The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed. My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli. 1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin. 2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017

abstract: Synthetic biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2018

Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-03-13T13:00:52Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Maria Paloma Silva de Barros_PPGG UFPE.pdf: 8103324 bytes, checksum: 8a69c263eb54cdd37eb3275985c8eb30 (MD5) / Made available in DSpace on 2015-03-13T13:00:52Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Maria Paloma Silva de Barros_PPGG UFPE.pdf: 8103324 bytes, checksum: 8a69c263eb54cdd37eb3275985c8eb30 (MD5) Previous issue date: 2012-09-14 / A peste é uma doença que permanece enraizada em inúmeros focos naturais por todo o mundo. Embora o Brasil passe por um período de silenciamento epidemiológico, anticorpos antipestosos são detectados nas atividades de vigilância, sugerindo que estes focos permanecem ativos. A Yersinia pestis, agente causador da peste, apresenta uma história evolutiva recente e é considerada uma espécie, geneticamente, muito homogênea. Diante da necessidade de estudos mais aprofundados sobre as características moleculares e evolutivas dos isolados de Y. pestis dos focos do Brasil, realizamos neste trabalho uma caracterização de cepas da coleção biológica (Fiocruz-CYP) de Yersinia, provenientes de cinco focos de peste do Brasil, com a finalidade de compreender a adaptação da bactéria no ambiente. Realizamos a padronização e a análise de macrorestrição (PFGE) em 22 cepas de Y. pestis, 17 de um surto ocorrido em 1986 e cinco isoladas da atividade de vigilância. O PFGE não separou os isolados da rotina e do surto, entretanto foi capaz de revelar diversidade genética entre as cepas. As técnicas MLVA e CRISPR também foram utilizadas na genotipagem das cepas de Y. pestis. Doze locos VNTR (MLVA) analisados em 37 cepas, pertencentes a dois eventos epidemiológicos distintos, permitiram observar a separação dos grupos por evento e estabelecer uma relação epidemiológica. Três locos CRISPR (YPa, YPb e YPc) foram analisados em 146 cepas, apenas dois (YPa e YPb) se mostraram polimórficos. A análise desta região permitiu realizar uma caracterização intraespecífica e microevolutiva dos isolados de peste dos focos brasileiros. O MLVA e o CRISPR demonstraram uma melhor relação entre os dados epidemiológicos e moleculares, enquanto o PFGE apenas diferenciou os isolados de Y. pestis. Os dados gerados pelas três técnicas estudadas permitiram confirmar que houve apenas a entrada de um clone de Y. pestis no Brasil e observar que alguns processos adaptativos foram necessários para sua interiorização e fixação nos focos do país.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T17:45:25Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-MariaPalomaBarros.pdf: 8071319 bytes, checksum: 0b3f7f14ac7d15cf16b3a5e185a6f89d (MD5) / Made available in DSpace on 2017-04-10T17:45:25Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-MariaPalomaBarros.pdf: 8071319 bytes, checksum: 0b3f7f14ac7d15cf16b3a5e185a6f89d (MD5) Previous issue date: 2012-09-14 / A peste é uma doença que permanece enraizada em inúmeros focos naturais por todo o mundo. Embora o Brasil passe por um período de silenciamento epidemiológico, anticorpos antipestosos são detectados nas atividades de vigilância, sugerindo que estes focos permanecem ativos. A Yersinia pestis, agente causador da peste, apresenta uma história evolutiva recente e é considerada uma espécie, geneticamente, muito homogênea. Diante da necessidade de estudos mais aprofundados sobre as características moleculares e evolutivas dos isolados de Y. pestis dos focos do Brasil, realizamos neste trabalho uma caracterização de cepas da coleção biológica (Fiocruz-CYP) de Yersinia, provenientes de cinco focos de peste do Brasil, com a finalidade de compreender a adaptação da bactéria no ambiente. Realizamos a padronização e a análise de macrorestrição (PFGE) em 22 cepas de Y. pestis, 17 de um surto ocorrido em 1986 e cinco isoladas da atividade de vigilância. O PFGE não separou os isolados da rotina e do surto, entretanto foi capaz de revelar diversidade genética entre as cepas. As técnicas MLVA e CRISPR também foram utilizadas na genotipagem das cepas de Y. pestis. Doze locos VNTR (MLVA) analisados em 37 cepas, pertencentes a dois eventos epidemiológicos distintos, permitiram observar a separação dos grupos por evento e estabelecer uma relação epidemiológica. Três locos CRISPR (YPa, YPb e YPc) foram analisados em 146 cepas, apenas dois (YPa e YPb) se mostraram polimórficos. A análise desta região permitiu realizar uma caracterização intraespecífica e microevolutiva dos isolados de peste dos focos brasileiros. O MLVA e o CRISPR demonstraram uma melhor relação entre os dados epidemiológicos e moleculares, enquanto o PFGE apenas diferenciou os isolados de Y. pestis. Os dados gerados pelas três técnicas estudadas permitiram confirmar que houve apenas a entrada de um clone de Y. pestis no Brasil e observar que alguns processos adaptativos foram necessários para sua interiorização e fixação nos focos do país. / Plague disease remains present in numerous natural foci worldwide. Although Brazil goes through a period of epidemiological silence antiplague antibodies are detected in the surveillance activities, suggesting that Brazilians foci remain active. Yersinia pestis, causative agent of plague, has a recent evolutionary history and is considered a very genetically homogeneous species. Given the need for further studies on the molecular and evolutionary characteristics of the isolates of Y. pestis of the Brazilian plague areas, we perform in this work a characterization of strains biological collection (Fiocruz-CYP) of Yersinia, from five outbreaks of plague in Brazil, with the aim of understanding the adaptation of bacteria to the environment. We carried out the standardization and macrorestriction analysis (PFGE) in 22 Y. pestis strains, 17 from an outbreak occurred in 1986 and five from surveillance activity. PFGE did not separate the isolates from surveillance and from outbreak, but it was able to reveal genetic diversity among strains. MLVA and CRISPR techniques were also used in genotyping Y. pestis strains. Analysis of 12 VNTR loci (MLVA) in 37 Y. pestisstrains, of two different epidemiological events, allowed observing the separation of groups establish an epidemiological link among the isolates. Three CRISPR loci (YPa, YPb and YPc) were analyzed in 146 Y. pestis strains, only two loci (YPa and YPb) were polymorphic. The analysis of this region allowed an intraspecific characterization and microevolutionary of the plague isolates in the Brazilian foci. The CRISPR and MLVA showed a better relationship between the epidemiological and molecular data, while PFGE differ only the Y. pestis strains. The data generated by the three techniques studied confirmed that there was only one entry clone Y. pestis in Brazil and observed that some adaptive processes were necessary for its internalization and fixation of these foci in our country.

PNPase is a gene implicated as a potential target for cancer therapy; human mutations also present with deafness, myopathies, and neuropathies. In this study, C. elegans was used to investigate the effect of knocking out PNPase in a whole animal. C. elegans knockdown studies have reported an extended lifespan via an increase in ROS production. Further noted are larger mitochondria and an increase in fzo-1 expression. Knockout animals previously constructed using CRISPR/Cas9 were used for this study. We aimed to confirm these findings validating previous studies. It was discovered that PNPase knockout animals demonstrated a similar lifespan extension that was resolved with the addition of antioxidants in the media. All subsequent findings contradicted those of the knockdown studies. Resequencing of knockout animals demonstrated no existing mutation and studies were discontinued. New mutants will advance future analyses and validate prior investigations.

Pluripotente Stammzellen haben das Potential, in unterschiedliche Zelltypen zu differenzieren und können genutzt werden, um organähnliche Mikrostrukturen zu generieren. Somit können molekulare Unterschiede verschiedenster künstlich differenzierter Gewebe, etwa zwischen Mensch und Schimpanse, anhand von pluripotenten Ausgangszellen untersucht werden. Da die Genome unserer nächsten ausgestorbenen Verwandten Neandertaler und Denisovaner aus konservierter DNA in alten Knochen sequenziert wurden, könnten ebenso Unterschiede zwischen Mensch und diesen Spezies oder dem letzten gemeinsamen Vorfahren untersucht werden. Dies erfordert jedoch die Generierung neandertalisierter Stammzellen durch künstliche Integration von Neandertalerallelen in humane Stammzellen, etwa durch die CRISPR Genomeditierungstechnik. Durch CRISPR kann ein DNA-Doppelstrangbruch an einer gewünschten Stelle im Genom eingefügt werden. Die zelluläre Reparatur des Doppelstrangbruchs ermöglicht dann die Editierung des Genoms. Basierend auf einer DNA-Matrize, die die gewünschte Modifikation trägt, kann das Genom an dieser Stelle präzise editiert werden. Die Effizienz präziser Editierung ist jedoch sehr niedrig im Vergleich zu unpräziser Reparatur. Um möglichst effizient neandertalisierte Stammzellen generieren zu können, wurden im Zuge dieser Doktorarbeit Methoden entwickelt, welche die präzise Genomeditierungseffizienz drastisch steigern. Zum einen wurde aus mehreren niedermolekularen Substanzen, welche mit Proteinen der DNA-Reparaturen interagieren, ein optimierter Mix entwickelt. Weiterhin konnte durch eine Mutation in einem zentralen Reparaturprotein die Effizienz für die Editierung eines einzelnen Gens auf 87% erhöht werden. Diese hohe Effizienz ermöglicht erstmals die präzise homozygote Editierung von vier Genen auf einmal in ein und derselben Zelle

Pre-mRNA splicing is the most critical process in gene expression regulation across eukaryotic species. This reaction is carried out by the spliceosome, a large, dynamic, and well-organized ribonucleoprotein complex. The spliceosome is composed of five major small nuclear RNAs and an excessive number of associated protein factors. Many protein splicing factors bind and release during splicing to assist the assembly and the modulation of many RNA structures and proteins within the spliceosome. CWC25 is a splicing protein factor that functions in modulating the conformational structure of the spliceosome at the first transesterification reaction. CWC25 binds with its N-terminus to the major groove of the catalytic spliceosome triggering the spliceosome activity. Here, we employed CRISPR/Cas9 genome engineering system for targeted mutagenesis to generate CWC25 functional knock-out mutants to understand its molecular function, contribution to splicing regulation and implication in fine-tuning responses to abiotic stress in rice. Our genotyping analysis of the OsCWC25 locus revealed the presence of two mono-allelic and 18 bi-allelic mutant lines. Phenotypic analysis of these mutants, including germination and root inhibition assays, showed that the cwc25 mutants are oversensitive to abiotic stresses such as ABA and salinity. Our data demonstrate that CWC25 plays an important role in regulating plant responses to abiotic stresses.