Einsatz eines modifizierten Cas9-Systems zur Promotorforschung im endogenen genomischen Kontext

Das Verständnis der komplexen Mechanismen der menschlichen Transkriptionsregulation bleibt trotz vollständiger Sequenzierung des Genoms eine große Herausforderung. Zur Erforschung wichtiger nicht kodierender DNA-Elemente stehen heute mehrere molekulargenetische Werkzeuge zur Verfügung. Unter Verwendung eines modifizierten Cas9-Komplexes entwickelten wir einen kombinierten experimentellen und modellierenden Ansatz zur Untersuchung cis-regulatorischer Interaktionen im nativen genomischen Kontext. Mit Hilfe einer katalytisch aktiven Cas9 haben wir das native humane Renin-Gen mit einem Firefly-Luziferase-kodierenden Gen stabil markiert, sodass die potenzielle Induktion der Genexpression durch ein optisches Signal messbar wurde. Für die anschließende experimentelle Arbeit fand eine modifizierte Cas9 Anwendung. Diese katalytisch inaktive Variante rekrutiert einen Komplex aus Transkriptionsfaktoren (SAM-Komplex), durch den die Expression eines gewünschten Zielgens induziert wird. Durch fünf designte Guide-RNAs konnten wir diesen SAM-Komplex zu spezifischen Promotorregionen des humanen Renins leiten. Die programmierten SAM-Komplexe induzierten in unseren Zellen die Aktivierung von Renin. Die resultierenden Genexpressionen wurden gemessen und in Computermodellen ausgewertet. Durch die Anwendung unseres kombinierten experimentellen und modellierenden Ansatzes können wir zeigen, dass relevante Interaktionen innerhalb der ausgewählten Sequenzen der proximalen Renin-Promotorregion auftreten. Unsere Ergebnisse zeigen, dass mit Hilfe von Weiterentwicklungen moderner molekulargenetischer Verfahren wie des Cas9-Systems und einem kombinatorischen, experimentellen und modellierenden Setup, eine suffiziente Promotorforschung im endogenen Kontext möglich wird

Ride the Tide: Observing CRISPR/Cas9 genome editing by the numbers

Targeted genome editing has become a powerful genetic tool for modification of DNA sequences in their natural chromosomal context. CRISPR RNA-guided nucleases have recently emerged as an efficient targeted editing tool for multiple organisms. Hereby a double strand break is introduced at a targeted DNA site. During DNA repair genomic alterations are introduced which can change the function of the DNA code. However, our understanding of how CRISPR works is incomplete and it is still hard to predict the CRISPR activity at the precise target sites. The highly ordered structure of the eukaryotic genome may play a role in this. The organization of the genome is controlled by dynamic changes of DNA methylation, histone modification, histone variant incorporation and nucleosome remodelling. The influence of nuclear organization and chromatin structure on transcription is reasonably well known, but we are just beginning to understand its effect on genome editing by CRISP

Quantification of CRISPR-Cas9 diffusion dynamics in Escherichia coli

What do adaptive immunity, genetic engineering and antimicrobials have in common? CRISPR-Cas9, the popular enzymatic complex that produces DNA double-strand breaks when associated with a guide-RNA. Hundreds of labs routinely use this system to edit genomic DNA; however, some of the mechanisms by which it interacts with nucleic acid remain unclear. In my lab, we developed an expertise in the study of DNA recombination, DNA repair and DNA interactions in Escherichia coli. We use single-molecule fluorescent microscopy to collect images in real time, in vivo. During my PhD, I harnessed this expertise to follow the behaviour of the Cas9 protein under different conditions: various expression levels; various gRNAs; and various genomic targets. By observing the diffusion dynamics of the protein, I was able to quantify how different DNA interactions were impacting the motion of the protein in the cytoplasm and inferred that actual ON-target interactions were very rare throughout the lifetime of the protein. In contrast, the protein was mainly involved in non-specific OFF-target DNA interactions, in search of its actual target. Additionally, my results reveal the presence of a large fraction of non-specific interactions, hitherto not reported in the literature, owing to their absence of DNA modification. In total, this work offers a collection of highly quantitative measurements on the behaviour of a protein whose activity is central to many biologists, while shedding a new light on the importance of Cas9 searching and targeting mechanisms. Finally, it opens a discussion on the role of DNA recognition in the context of gene editing and antimicrobial resistance

Mining and characterization of the candidate genes for distorted segregation in chromosome 4 of tomato

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em PlantasSolanum lycopersicum L. genetic variability was drastically diminished by successive genetic bottlenecks induced by the domestication process. The wild species of tomato, S. pimpinellifolium, is a small red-fruited plant native to Peru and is assumed as an ancestor species of the domesticated S. lycopersicum. One of the strategies to induce genetic variability to cultivated tomato is the development of introgression lines (ILs) containing a single segment of a donor wild genome in the genetic background of an elite tomato cultivar. In 2014, a genomic library of ILs that incorporates variability from the S. pimpinellifolium accession TO-937 in the genetic background of S. lycopersicum cultivar “Moneymaker” was developed. During the development of the IL collection, a region on the distal portion of chromosome 4 showed a segregation distortion (SD) favouring TO-937 alleles in detriment of “Moneymaker” alleles. Recently, the SD region was mapped to a 39Kb region of chromosome 4 containing seven gene annotations. The preliminary studies to assert gametic, post gametic and/or zygotic indicated that the SD was most probably caused by post-gametic or zygotic selection and it was a sex-independent phenomenon. The present study aims to characterize the genes included in the SD region and to propose a possible mechanism for the SD. Expression profile analysis by qRT-PCR and sequencing of genomic and transcriptomic sequences indicated a strong expression in the reproductive tissues of the two Heat-Shock Protein (HSP) genes contained in the SD region. Haplotyping of reciprocal and self-pollinating crosses between the SD haplotypes and “Moneymaker” gave new insights about the gametic and zygotic character of the SD. The analysis of natural sequence variations of the SD region revealed this region diverged in wild tomato accessions. Additionally, a reverse genetic approach was initated to assess if the HSPs are the cause of the SD using the GoldenBraid 3.0 standard assembly to create Agrobacterium-mediated transformation vectors, two CRISPR/Cas9 expression cassettes for the silencing of the HSP genes, and 3 expression cassettes.A variabilidade genética de Solanum lycopersicum L. foi drasticamente diminuída por sucessivos efeitos de gargalo genéticos induzidos pelo processo de domesticação. A espécie de tomate selvagem, S. pimpinellifolium, é uma pequena planta de frutos vermelhos nativas do Peru e é assumida como um ancestral do S. lycopersicum domesticado. Uma das estratégias para induzir variabilidade genética ao tomate cultivado é o desenvolvimento de linhas de introgressão (ILs - introgression lines) que contêm um único segmento do genoma selvagem no genoma de um cultivar de elite. Em 2014, foi desenvolvida uma biblioteca genómica de ILs que incorpora variabilidade da acessão TO-937 de S. pimpinellifolium no genoma do cultivar "Moneymaker" de S. lycopersicum. Durante o desenvolvimento da IL, uma região na porção distal do cromossoma 4 revelou uma segregação distorcida (SD) favorecendo os alelos TO-937 em detrimento dos alelos "Moneymaker". Recentemente, a região SD foi mapeada para uma região de 39Kb do cromossoma 4 contendo sete genes. Os estudos preliminares para afirmar o caracter gamético, pós-gamético e/ou zigótico indicaram que a SD provavelmente é causada pela seleção pósgamética ou zigótica e é um fenómeno independente do sexo. O presente estudo tem como objetivo a caracterização dos genes incluídos na região SD e um possível mecanismo de SD. A análise de perfil de expressão por qRT-PCR e sequenciação do genoma e transcriptoma indicou uma elevada expressão em tecidos reprodutores de dois genes contidos na região genómica da SD que codificam Heat-Shock Proteins (HSP). Haplotipagem de cruzamentos recíprocos e autocruzamentos entre os haplótipos de SD e "Moneymaker" revelou novas pistas acerca do caráter gamético e zigótico da SD. A análise da variação natural da região SD revelou uma significativa diversidade em acessões de tomate selvagem. Além disso, usando o sistema de clonagem GoldenBraid 3.0 para criar vetores de transformação mediada por Agrobacterium, duas cassetes de expressão CRISPR / Cas9 para o silenciamento dos genes HSP e 3 cassetes de expressão para foram desenvolvidas para futura aplicação

Automation-aided high-throughput technologies for synthetic biology

Synthetic biology is a research discipline which harnesses technological progress in de novo DNA synthesis as well as combining expertise of biological sciences and engineering research fields to facilitate construction of novel artificial biological systems. Since the past two decades, application of its methodologies has led to significant advances in metabolic engineering, providing alternative biochemical routes for the production of therapeutic products, cosmetics and biofuels. However, several challenges remain to be addressed to support development of synthetic biology applications, notably the demand for faster, cheaper and more reliable DNA manufacturing as well as efficient methods for genome-scale engineering of living organisms. This doctoral thesis proposes new interdisciplinary approaches to these problems, taking advantage of the latest laboratory automation technologies to improve efficiency of modern DNA assembly and genome editing methods. The first results chapter proposes application of a robotic platform for an acoustic liquid transfer for miniaturisation of DNA fabrication. This research, published in 2016, demonstrates the possibility to cost-efficiently assemble DNA in sub-microlitre assembly reactions. The second results chapter presents efforts to develop a method for genome-scale engineering of a model eukaryote, the budding yeast. This work capitalises on the recent progress in on-chip DNA synthesis and the next-generation sequencing (NGS) technology. Finally, the last results chapter demonstrates computational studies to predict and accelerate turnaround times of a commercial DNA supply chain using probabilistic simulations. The developed software is used to estimate sequence-specific DNA manufacturing turnaround times in order to help plan DNA manufacturing and guide decisions regarding further automation of different experimental procedures

CRISPR/Cas9-based strategies for pig host resistance to Influenza A virus

Influenza circulates in different mammalian and avian species, causing epidemics and occasional pandemics. This poses a substantial threat to agricultural productions, animal welfare, human public health, and economy. The causative agent of the disease is Influenza A virus (IAV), whose entry depends on its preference on the receptor molecules since the preference determines whether virus glycoproteins can employ the host cell surface sialic acid (SA) as ligands. There are two major types of sialylated glycans as receptors for viral recognition: one is Neu5Ac α 2,6-Gal (SA α 2,6-Gal for short) preferentially recognised by human IAVs, and the other one is Neu5Ac α 2,3-Gal (SA α 2,3-Gal for short) predominantly recognised by avian IAVs. Pigs harbouring both types of SA-containing receptors have the potential to play a role of ‘intermediate hosts’ or ‘mixing vessels’. Therefore, it is of great interest to develop methods aiming at reducing α 2,6-SA-containing receptors in pig cells, so that they are less susceptible to human IAV infections. β-galactoside α 2,6-sialyltransferase 1 (ST6Gal1) mediates N-linked α 2,6- sialylation on cell surfaces by catalysing the addition of α 2,6-SA to the terminal N-glycans. ST6Gal1 is involved in a wide range of biological events, such as the generation of carbohydrate determinants on the cell surfaces, the immune regulation, and in various carcinomas. ST6Gal1 is encoded by the ST6GAL1 gene, which expression has been reported to display a tissue-specific pattern as a result of the regulations of multiple promoter regions and the differential combinations of 5’- untranslated exons. Driven by the concern that inactivating the ST6GAL1 gene may have deleterious phenotypic effects given the widespread expression profile and the diversity of its biological functions, I pursued a subtle approach to engineering the ST6GAL1 gene — that of removing a single promoter region to alter the expression profile. I hypothesised that reducing the biosynthesis of α 2,6-sialylated glycan structure exclusively on the respiratory tract could potentially block IAV entry without compromising humoral immune responses. To this end, I identified 5’ transcription starting sites (TSSs) and 5’ untranslated region (UTR) exons of transcripts expressed in nine pig tissues. Then we employed the CRISPR/Cas9 system to precisely engineer pig ST6GAL1 gene instead of the whole gene deletion. The consequence of deleting the region surrounding the 5’ TSS of ST6GAL1 transcripts predominantly expressed in airway was assessed (the resulting model was termed as ST6GAL1ΔP). Moreover, I generated a ST6GAL1 functional knockout model by inducing a frameshift mutation in pig trachea cells (the resulting model was termed as ST6GALΔCD). Additionally, human IAV had reduced infectivity in ST6GAL1ΔP relative to non-edited cells, suggesting that a strategy to reduce the biosynthesis of α 2,6-sialylated glycan structure exclusively on the airway could offer an antiviral strategy, independent of inducing a humoral immune response. This work lays a solid foundation in generating engineered pigs for IAV host resistance modelling, and helps us to achieve the genetic improvement in swine herds; also, it provides a good understanding of the fundamental molecular basis of the IAV-host interactions, and develop novel antiviral and therapeutics strategies

Yeast as a platform for synthetic biology and investigation of evolutionary hypotheses

Yeast has long been the sine qua non of model organisms due to its experimental tractability. Recent advances in biology, such as CRISPR/Cas9 editing, promise to bring this tractability to other model organism. But the same advances have also added new dimensions to the utility of yeast, and reinforced its importance as a unique platform for studying basic biology, translational research and building the future of biotechnology. In this thesis I describe (i) the use of humanized yeast to study fundamental questions about the evolution of genetic systems, (ii) rapid and versatile techniques for leveraging classical yeast techniques along with cutting edge developments to solve 21-st century problems and (iii) a blueprint for transforming yeast into a vessel for storage of digital information.Cellular and Molecular Biolog

MUrine tools to catch high-affinity plasma cells (MUTCHAP)

The generation of high-affinity antibodies is critical for natural and induced protective immune responses and production of efficacious therapeutic monoclonal antibodies. Germinal centre matured plasma cells (GCmat PCs) produce these long-term high-quality responses. The signals and mechanisms by which GCmat PCs are induced are not fully elucidated. We developed parallel strategies to generate novel gene manipulated mice where GCmat PCs are easily identifiable by fluorescent markers controlled by GC-specific S1pr2 and PC-specific Prdm1 expression. Combination of validated transgenic alleles generated the High-Affinity LOw-affinity (HALO) PC mouse, able to distinguish between GCmat PCs and extrafollicular PCs. Kinetic assessment of early GC output confirmed previous results from our lab that GCmat PC output peaks early then declines. Furthermore, we detected transient re-expression of CD38 in GCmat PC precursors and GCmat transitioning PCs, an intriguing result with a yet undetermined function. In tandem, we developed and characterised a novel split fluorescent reporter, diSplit670. CRISPR/Cas9 enhanced targeted insertion placed each part of diSplit670 under control of endogenous Prdm1 and S1pr2 in separate mouse embryonic stem cell (ESC) lines. To-date, germline transmission has been achieved for one targeted ESC line. These novel tools would be uniquely suited to interrogate the biology of GCmat PCs

Deep mutational scanning of mammalian loci using CRISPR-Cas9 and multiplex HDR

Functional consequences of genetic variants are best studied in their endogenous chromosomal context. Gene editing by homology-directed repair can introduce such predetermined genetic changes into chromosomal DNA. In this thesis, I develop methods to generate tens to hundreds of genetic variants, expressed from a native chromosomal context, and simultaneously evaluate their phenotypic impact. This approach involves repair of Cas9-derived double strand breaks (DSBs) from oligonucleotide repair template libraries containing controlled levels of nucleotide heterogeneity. Cell populations are then purified based on a phenotypic assay and subjected to deep amplicon sequencing at the target site to link genotype with phenotype. In the first chapter, I developed a bioinformatics pipeline for the processing of Illumina sequencing reads containing nucleotide variants, and validate this pipeline in silico. As a proof-of-principle, in the second chapter I then introduced nucleotide variants across 8 codons of a chromosomal GFP transgene in mouse embryonic stem cells. The functional impact of these variants was quantified, with the results benchmarked against an existing episomal dataset, and by in silico modelling of mutant protein structure. In the final chapter, I applied this pipeline to analyse a CRISPR deep mutational scanning dataset incorporating all possible amino acid substitutions within a region of β-catenin, a component of the Wnt signalling pathway, that is a mutational hotspot in many types of cancer. The functional impact of these clinically relevant variants was assessed using a fluorescent reporter of Wnt signalling. By combining the resulting functional scores with mutational signature data from genome sequencing of different tumour types, I finally dissect the relative contribution of mutational bias and natural selection to the different patterns of amino acid substitutions found in different tumour types