Development of CRISPR-based programmable transcriptional regulators and their applications in plants

[ES] La Biología Sintética de Plantas tiene como objetivo rediseñar las plantas para que adquieran características y funcionalidades novedosas a través de circuitos reguladores ortogonales. Para lograr este objetivo, se deben desarrollar nuevas herramientas moleculares con la capacidad de interactuar con factores endógenos de manera potente y específica. CRISPR/Cas9 surgió como una herramienta prometedora que combina la capacidad personalizable de unión al DNA, a través de la versión catalíticamente inactivada de la proteína Cas9 (dCas9), con la posibilidad de anclar dominios autónomos de activación transcripcional (TADs) a su estructura para lograr una regulación específica de la expresión génica. Los activadores transcripcionales programables (PTAs) pueden actuar como procesadores específicos, ortogonales y versátiles para el desarrollo de nuevos circuitos genéticos en las plantas. En busca de dCas9-PTA optimizados, se llevó a cabo una evaluación combinatoria de diferentes arquitecturas dCas9 con un catálogo de varios TAD. La mejor herramienta resultante de esta comparación, denominada dCasEV2.1, se basa en la estrategia scRNA y la combinación de los dominios de activación EDLL y VPR con un bucle multiplexable gRNA2.1, que es una versión mutada del gRNA2.0 descrito previamente. En este trabajo, el activador programable dCasEV2.1 demostró ser una herramienta potente y específica, logrando tasas de activación más altas que otras estrategias dCas9 disponibles en plantas. Se observaron tasas de activación sin precedentes dirigidas a genes endógenos en N. benthamiana, acompañadas de una estricta especificidad en todo el genoma, lo que hace que esta herramienta sea adecuada para la regulación estricta de redes reguladoras complejas. Como prueba de concepto, se diseñaron cuatro programas de activación para distintas ramas de la ruta de los flavonoides, buscando obtener enriquecimientos metabólicos específicos en hojas de N. benthamiana. El análisis metabólico de las hojas metabólicamente reprogramadas mediante dCasEV2.1 reveló un enriquecimiento selectivo de los metabolitos diana y sus derivados glicosilados, que se correlacionaron con el programa de activación empleado. Estos resultados demuestran que dCasEV2.1 es una herramienta eficaz para la ingeniería metabólica y un componente clave en los circuitos genéticos destinados a reprogramar los flujos metabólicos. Finalmente, basándonos en dCasEV2.1, desarrollamos un sistema optimizado de regulación de genes inducidos por virus (VIGR) que utiliza un vector Potato Virus X (PVX) para el suministro de los programas de activación CRISPR codificados con gRNA. Este enfoque permite controlar el transcriptoma de la planta a través de una aplicación sistémica basada en aerosol de componentes CRISPR a plantas adultas. El nuevo sistema PVX-VIGR produjo una fuerte activación transcripcional en varios genes diana endógenos, incluidos tres factores de transcripción MYB-like seleccionados. Las activaciones específicas de MYB condujeron a perfiles metabólicos distintivos, demostrando que las aplicaciones potenciales de la herramienta dCasEV2.1 en plantas incluyen la obtención de perfiles metabólicos personalizados utilizando un suministro basado en aerosol de instrucciones de reprogramación transcripcional codificadas por gRNA. En resumen, esta tesis proporciona herramientas novedosas para la activación transcripcional fuerte, ortogonal y programable en plantas, con una caja de herramientas ampliada para el suministro de los programas de activación.[CA] La Biologia Sintètica de Plantes té com objectiu redissenyar les plantes per que obtinguen característiques i funcionalitats innovadores mitjançant circuits reguladors ortogonals. Per arribar a aquest objectiu, s'han de desenvolupar noves ferramentes moleculars amb la capacitat d'interactuar amb factor endògens d'una manera potent i específica. CRISPR/Cas9 va sorgir com una ferramenta prometedora que combina la capacitat personalitzable d'unió al DNA, mitjançant la versió catalíticament inactivada de la proteïna Cas9 (dCas9), amb la possibilitat de fixar dominis autònoms de activació transcripcional (TADs) a la seua estructura per aconseguir una regulació específica de la expressió gènica. Els activadors transcripcionals programables (PTAs) poden actuar com a processadors específics, ortogonals i versàtils per al desenvolupament de nous circuits genètics a les plantes. Buscant dCas9-PTA optimitzats, es va realitzar una avaluació combinatòria de distintes arquitectures dCas9 amb un catàleg de diversos TAD. La millor ferramenta segons aquesta comparació, anomenada dCasEV2.1, es basa en la estratègia scRNA i la combinació del dominis d'activació EDLL i VPR amb un bucle multiplexable gRNA2.1, que es una versió mutada del gRNA2.0 descrit prèviament. En aquest treball, el activador programable dCasEV2.1 es va mostrar com una ferramenta potent i específica, aconseguint nivells d'activació majors que altes estratègies dCas9 disponibles en plantes. Es van observar taxes d'activació sense precedents dirigides a gens endògens en N. benthamiana, junt a una estricta especificitat en tot el genoma, indicant que aquesta ferramenta és adequada per a la regulació estricta de xarxes reguladores complexes. Como proba de concepte, se van dissenyar quatre programes d'activació per a diferent branques de la ruta dels flavonoides, cercant obtenir enriquiments metabòlics específics en fulles de N. benthamiana. L'anàlisi metabòlic de les fulles metabòlicament reprogramades mitjançant dCasEV2.1 va revelar un enriquiment selectiu del metabòlits diana i els seus derivats glicosilats que es correlacionen amb el programa d'activació emprat. Aquests resultats demostren que dCasEV2.1 és una ferramenta eficaç per a l'enginyeria metabòlica i un component clau als circuits genètics destinats a reprogramar els fluxos metabòlics. Finalment, en base a dCasEV2.1, desenvoluparem un sistema optimitzat de regulació de gens induïts per virus (VIGR) que utilitza un vector Potato Virus X (PVX) per al subministrament dels programes d'activació CRISPR codificats amb gRNA. Aquesta aproximació permet controlar el transcriptoma de la planta mitjançant l'aplicació sistèmica basada en aerosol de components CRISPR a plantes adultes. El nou sistema PVX-VIGR va produir una gran activació transcripcional en diversos gens diana endògens, inclosos tres factors de transcripció MYB-like seleccionats prèviament. Les activacions específiques de MYB conduïren a perfils metabòlics distintius, demostrant que les aplicacions potencials de la ferramenta dCasEV2.1 en plantes inclouen la obtenció de perfils metabòlics personalitzats emprant un subministrament basat en aerosol de instruccions de reprogramació transcripcional codificades per gRNA. En resum, aquesta tesis proporciona noves ferramentes per a l'activació transcripcional forta, ortogonal i programable en plantes, amb una caixa de ferramentes eixamplada per al subministraments dels programes d'activació.[EN] Plant Synthetic Biology aims to redesign plants to acquire novel traits and functionalities through orthogonal regulatory circuits. To achieve this goal, new molecular tools with the capacity of interacting with endogenous factors in a potent and specific manner must be developed. CRISPR/Cas9 emerged as promising tools which combine a customizable DNA-binding activity through the catalytically inactivated version of Cas9 protein (dCas9) with the possibility to anchor autonomous transcriptional activation domains (TADs) to its structure to achieve a specific regulation of the gene expression. The Programmable Transcriptional Activators (PTAs) could act as specific, orthogonal and versatile processor components in the development of new genetic circuits in plants. In search for optimized dCas9-PTAs, a combinatorial evaluation of different dCas9 architectures with a catalogue of various TADs was performed. The best resulting tool of this comparison, named dCasEV2.1, is based on the scRNA strategy and the combination of EDLL and VPR activation domains with a multiplexable gRNA2.1 loop, which is a mutated version of the previously described gRNA2.0. In this work, the dCasEV2.1 programable activator was proved to be a strong and specific tool, achieving higher activation rates than other available dCas9 strategies in plants. Unprecedented activation rates were observed targeting endogenous genes in N. benthamiana, accompanied by strict genome-wide specificity that makes this tool suitable to perform a tight regulation of complex regulatory networks. As a proof of concept, a design of four activation programs to activate different branches of the flavonoid pathway and obtain specific metabolic enrichments in N. benthamiana leaves was performed. The metabolic analysis on the dCasEV2.1 metabolically reprogrammed leaves revealed a selective enrichment of the targeted metabolites and their glycosylated derivatives that correlated with the activation program employed. These results demonstrate that dCasEV2.1 is a powerful tool for metabolic engineering and a key component in genetic circuits aimed at reprogramming metabolic fluxes. Finally, based on dCasEV2.1, we developed an optimized Viral Induced Gene Regulation (VIGR) system that makes use of a Potato Virus X (PVX) vector for the delivery of the gRNA-encoded CRISPR activation programs. This approach offers a way to control the plant transcriptome through a spray-based systemic delivery of CRISPR components to adult plants. The new PVX-VIGR system led to strong transcriptional activation in several endogenous target genes, including three selected MYB-like transcription factors. Specific MYB activations lead to distinctive metabolic profiles, showing that the potential applications of the dCasEV2.1 tool in plants include the obtention of custom metabolic profiles using a spray-based delivery of gRNA-encoded transcriptional reprogramming instructions. In sum, this thesis provides novel tools for strong, orthogonal and programmable transcriptional activation in plants, with an expanded toolbox for the delivery of the activation programs.Selma García, S. (2022). Development of CRISPR-based programmable transcriptional regulators and their applications in plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185046TESI

MAPPING LOW-FREQUENCY FIELD POTENTIALS IN BRAIN CIRCUITS WITH HIGH-RESOLUTION CMOS ELECTRODE ARRAY RECORDINGS

Neurotechnologies based on microelectronic active electrode array devices are on the way to provide the capability to record electrophysiological neural activity from a thousands of closely spaced microelectrodes. This generates increasing volumes of experimental data to be analyzed, but also offers the unprecedented opportunity to observe bioelectrical signals at high spatial and temporal resolutions in large portions of brain circuits. The overall aim of this PhD was to study the application of high-resolution CMOS-based electrode arrays (CMOS-MEAs) for electrophysiological experiments and to investigate computational methods adapted to the analysis of the electrophysiological data generated by these devices. A large part of my work was carried out on cortico-hippocampal brain slices by focusing on the hippocampal circuit. In the history of neuroscience, a major technological advance for hippocampal research, and also for the field of neurobiology, was the development of the in vitro hippocampal slice preparation. Neurobiological principles that have been discovered from work on in vitro hippocampal preparations include, for instance, the identification of excitatory and inhibitory synapses and their localization, the characterization of transmitters and receptors, the discovery of long-term potentiation (LTP) and long-term depression (LTD) and the study of oscillations in neuronal networks. In this context, an initial aim of my work was to optimize the preparation and maintenance of acute cortico-hippocampal brain slices on planar CMOS-MEAs. At first, I focused on experimental methods and computational data analysis tools for drug-screening applications based on LTP quantifications. Although the majority of standard protocols still use two electrodes platforms for quantifying LTP, in my PhD I investigate the potential advantages of recording the electrical activity from many electrodes to spatiotemporally characterized electrically induced responses. This work also involved the collaboration with 3Brain AG and a CRO involved in drug-testing, and led to a software tool that was licensed for developing its exploitation. In a second part of my work I focused on exploiting the recording resolution of planar CMOS-MEAs to study the generation of sharp wave ripples (SPW-Rs) in the hippocampal circuit. This research activity was carried out also by visiting the laboratory of Prof. A. Sirota (Ludwig Maximilians University, Munich). In addition to set-up the experimental conditions to record SPW-Rs from planar CMOS-MEAs integrating 4096 microelectrodes, I also explored the implementation of a data analysis pipeline to identify spatiotemporal features that might characterize different type of in-vitro generated SPW-R events. Finally, I also contributed to the initial implementation of high-density implantable CMOS-probes for in-vivo electrophysiology with the aim of evaluating in vivo the algorithms that I developed and investigated on brain slices. With this aim, in the last period of my PhD I worked on the development of a Graphical User Interface for controlling active dense CMOS probes (or SiNAPS probes) under development in our laboratory. I participated to preliminary experimental recordings using 4-shank CMOS-probes featuring 1024 simultaneously recording electrodes and I contributed to the development of a software interface for executing these experiments

A Serial Multiplexed Approach to Immunolabeling Brain Tissue for Electron Microscopy

To fully understand how biological systems give rise to complex phenomena, both structural and functional knowledge of the components in the system must be acquired. In this manuscript, I explain why new methods must be developed to achieve this level of understanding with brain tissue and introduce serial multiplexing as a potential solution. I discuss how this method can lead to highly annotated volume reconstructions of brain tissue using electron and light microscopy, along with the steps required to do so. I present what our lab has found throughout the process of adapting, refining, and combining more traditional methods together to work harmoniously for achieving multiplexed labeling of brain tissue. I also discuss what methods we found to be incompatible and what our advice is for others looking to achieve similar results. I explain why electron microscopy is required to extract ultrastructural information of brain tissue such as synapses, dendritic morphology, and subcellular components. Topics including sample preservation and storage, serial sectioning, grid handling, and immunohistochemistry, among others, are also discussed. To perform immunohistochemistry on brain tissue without compromising its structural integrity in the electron microscope is a considerable challenge. This challenge is magnified when the goal is to label an unrestricted number of target proteins in a sample destined for volume reconstruction, especially considering the lack of a unified roadmap for doing so. We therefore conducted experiments to test the efficacy and reliability of established techniques throughout all processes of obtaining brain tissue for serial multiplexed electron microscopy. We also tested the efficacy and dilution requirements of antibodies across several parameters, targeting candidate proteins and molecules of interest using immunofluorescence microscopy. We draw preliminary conclusions regarding our findings and explore possible directions for the project to continue in the future. The work presented in this manuscript reflects the concerted effort of many individuals over a year and a half and was unfortunately forced to stop prematurely due to a public health crisis beyond our control

Online and Distance Learning during Lockdown Times: COVID-19 Stories (Volume 1)

This book is a reprint of papers in the Special Issue published in Education Sciences under the title "Online and Distance Learning during Lockdown Times: COVID-19 Stories". It includes papers covering K-12 educational sector representing international experience of teaching and learning from the start of the first episode of lockdown due to the Covid-19 pandemic

Liquid biopsies in pediatric oncology : towards clinical implementation

During the last few years, liquid biopsies based on cell-free DNA (cfDNA) have become approved for clinical use in several areas of adult oncology. Due to the minimally invasive nature of this procedure, liquid biopsies could serve many purposes in childhood oncology as well. For instance, the method can provide highly specific biomarkers for molecular diagnostics, enable identification therapeutic targets and, more frequently, be used to assess therapeutic response or disease recurrence. This information could aid clinical decision-making in tailoring treatment and follow-up for each individual patient. However, developing customized assays for children's cancers is demanding for several reasons. First, childhood cancer is rare compared to adult cancers, making it difficult to get the numbers needed for statistically powered studies. Second, children’s cancers generally have few and often private mutations, and display a different mutational landscape than adult counterparts, meaning that generic assays are very challenging to develop. Third, small children mean small liquid biopsy volumes and high risk for subsampling errors, demanding ultrasensitive techniques for disease detection. Due to these circumstances, liquid biopsy assays for children with cancer require some innovative strategies. In this thesis, we describe a few liquid biopsy pilot studies focusing on the two main groups of childhood cancers; acute lymphoblastic leukemia and central nervous system (CNS) tumors. In paper I, we use whole genome sequencing to identify structural variants (SVs) in leukemic cells and use the resulting unique sequences as targets for patient-specific droplet digital PCR (ddPCR) assays. Based on analysis of samples from six children, we show that it is technically feasible to use this approach to accurately quantify measurable residual disease in bone marrow as well as in cfDNA from plasma. The molecular assays also enabled detection of potential low-grade CNSinvolvement in half of patients at diagnosis. In paper II, we use a similar approach analyzing samples from 12 children with medulloblastoma, this time combining single nucleotide variants and SVs in our assays. We show that post-operatively, all tumors that grew in contact with cerebrospinal fluid (CSF) prior to resection, and where imaging supported or could not rule out residual tumor after surgery, molecular signs of the disease were seen in liquid biopsies on at least one occasion within three weeks of surgery. Furthermore, most plasma samples at diagnosis also bore molecular signs of the tumors. In paper III, we analyze cfDNA in CSF from a child with an inoperable brainstem tumor for BRAF V600E/K/R mutations. After confirmation of a BRAF mutation, molecular targeted therapy was initiated with dramatic clinical response during the first nine months of treatment. In paper IV, we used multiomics data to seek a molecular diagnosis for a child born with a large CNS tumor. The data revealed a rare type of glioma, most likely caused by a novel fusion gene; SNRNP70::ALK. Molecular targeted therapy was initiated and four months into treatment, there are clinical and radiological signs of treatment effect. Taken together, the results indicate that our approach to design molecular assays could expand the utility of ddPCR for liquid biopsy analysis. Furthermore, in clinically challenging cases, multiomics can resolve complex diagnoses and liquid biopsies can guide choice of treatment. These studies are the initial efforts of our research group to promote liquid biopsies as a powerful precision diagnostic tool in pediatric oncology. Apart from the 20 patients described in these studies, another 130 children have been included for research and a liquid biopsy biobank of >1800 samples has been established. We have also set up clinical liquid biopsy analysis for BRAF V600 and H3-3A K27M mutations during this period. This was achieved through a close collaboration between the Departments of Clinical Genetics, Pediatric Oncology, Pediatric Neurosurgery, Clinical Pathology and The Swedish Childhood Tumor Biobank