CRISPR/Cas9 system optimization in Pseudomona syringae using a heterologous repair system

Motivation: The methods of genetic manipulation in most Pseudomonas species, such as Pseudomonas syringae, remain a laborious endeavor. However, the application of the CRISPR-Cas9 system in this bacterium would allow a means of fast and directed genetic edition. The CRISPR-Cas9 system produces a doubles-stranded DNA break by the Cas9 protein. This break can be repaired by the NHEJ machinery in a very low frecuence either by the homologous repair system, which is widely spread amoung prokaryotes. The bacteria Pseudomona syringae pv. phaseolicola and pv. tomato are phytopathogenic causing diseases that affect crops of high economic value. Therefore, the optimization of a genetic modification system for these bacteria is very important. Methods: In this study we report the development of the CRISPR Cas9 technology in Pseudomonas syringae pv tomato and pv. phaseolicola by using through the transformation of this bacteria with standardized pSEVA plasmids. After incorporating, the plasmids expressing the Cas9 nuclease and the guide RNA (gRNA), the Cas9 protein is complexed with the gRNA designed to direct a double-strand break to the pyrF gene. This lesion might be repaired by the bacterium intrinsic repair systems, but with a very low frequency. In order to solve this problem, a plasmid family has been used for expression in Pseudomonas syringae of three bacterial NHEJ systems from Sphingopyxis granuli, Mycobacterium tuberculosis and Mycobacterium smegmatis. Results: The results shown in this study provide a step ahead towards the genetic modification in Gram negative bacteria by using the CRISPR Cas9 system and a non homologos end joining system together. Nevertheless it should be emphasised that the Cas9 protein can be toxic in this bacteria producing non-targeted mutations, because of that is very important keep the nuclease cas9 as little time as possible in the bacteriu

Chromatin regulation of virulence gene clusters in Ustilago maydis.

Pathogenic fungi possess chromatin modification mechanisms that are involved in their virulence and interaction with the host 1. This modification usually occurs as a consequence of the presence or absence of certain molecular marks on histones, such as methylations or acetylations. It allows the transition between their two conformational states, euchromatin and heterochromatin, associated with gene transcriptional activation and gene silencing, respectively 2,3. The maize fungus Ustilago maydis has become a model organism for the study of plant pathogenesis due to its ease of handling and the multiple available genetic and cellular tools 4. This pathogen uses a large number of factors and pathways that regulate gene expression at each stage of the infective process, but little is known about the role of chromatin in that 1. Many genes involved in that process are clustered in gene regions and are activated at specific times 4,5. Therefore, we should think of an active role of chromatin in this regulation. However, the absence of typical heterochromatin-associated marks and regulators in U. maydis has so far been observed 1. In this laboratory, we have sought other likely regulators of this process and found that the histone methyltransferase Ash1 affects the virulence activity through the silencing of gene clusters involved in this process. In this project we try to find out if this is the only regulator of heterochromatin in U. maydis. For this purpose, different strains with antibiotic resistance markers in silenced regions controlled by Ash1 were developed, allowing antibiotic resistance when these regions are derepressed. Spontaneous resistant mutants were obtained from these strains and Ash1 was consequently sequenced to determine if the silencing was affected by a mutation in Ash1. Surprisingly, the methyltransferase gene was not mutated, suggesting the presence of other regulators. In addition, we performed a ChIP-qPCR to see if the epigenetic mark associated with Ash1 was still present. We observed a decrease in this mark in one of the spontaneous mutants in different virulence regions, which also suggests the presence of other regulators, such as some demethylases. Subsequently, in order to discover whether the decrease in this mark is maintained over time, we have studied its stability in successive generations. We noticed antibiotic-resistant colonies trended towards a decrease which might imply a reacquisition of the epigenetic mark

Desarrollo de una herramienta para evaluar la eficiencia de reparacio¿n de cortes de doble cadena por sistemas de NHEJ

Trabajo presentado en el XXVIII Congreso Nacional de Microbiología de la SEM, celebrado en modalidad virtual del 28 de junio al 02 de julio de 2022

Desarrollo de un sistema de edición genómica CRISPR-Cas de aplicación generalizada en bacterias Gram-negativas

Resumen del trabajo presentado en la XIII Reunión del grupo especializado en Microbiología Molecular de la SEM, celebrada en Granada (España) del 07 al 09 de septiembre de 2022.La rápida emergencia de las herramientas de edición genética basados en los sistemas de inmunidad adquirida CRISPR-Cas presentes en un buen número de bacterias y arqueas ha representado la mayor revolución de la última década en lo que respecta a la manipulación genética dirigida de una gran variedad de organismos. A pesar de que entre las bacterias Gram-negativas se encuentra una inmensa variedad de organismos de gran interés biológico, clínico, ambiental y biotecnológico, los métodos avanzados de edición genómica solo se han aplicado en un número limitado de especies. Con objeto de facilitar la implantación de esta metodología en un número amplio de bacterias Gram-negativas con mínimos requerimientos, hemos desarrollado una plataforma, denominada DEMOCRISPR, compuesta de una colección de vectores plasmídicos de amplio espectro para la producción de (i) las proteínas Cas de distintos sistemas CRISPR-Cas, (ii) gRNAs específicos compatibles con dichos sistemas, (iii) sistemas de reparación mediante empalme no homólogo de extremos (NHEJ), y (iv) sistemas de reparación dirigida por recombinación (HDR) de diversos orígenes. En paralelo, hemos construido una serie de vectores plasmídicos con reporters fluorescentes para cuantificar la eficiencia de los procesos de corte de doble cadena y reparación mutagénica. El uso de plásmidos de amplio espectro de hospedador compatibles entre sí para la producción de los complejos endonucleasa CRISPR y los sistemas de reparación, acompañados de espaciadores y secuencias diana estandarizados en reporters fluorescentes facilita la exportación de la plataforma a diversas bacterias Gram-negativas sin necesidad de diseñar gRNAs específicos para dianas presentes en cada organismo. Como prueba de principio, en este trabajo presentamos resultados que muestran la implementación de la plataforma DEMOCRISPR en distintas bacterias Gram-negativas.Este trabajo está financiado por el Programa Operativo FEDER Andalucía, convocatoria 2018 (proyecto UPO-1264127; IP: Fernando Govantes

Discovering HIV related information by means of association rules and machine learning

Acquired immunodeficiency syndrome (AIDS) is still one of the main health problems worldwide. It is therefore essential to keep making progress in improving the prognosis and quality of life of affected patients. One way to advance along this pathway is to uncover connections between other disorders associated with HIV/AIDS-so that they can be anticipated and possibly mitigated. We propose to achieve this by using Association Rules (ARs). They allow us to represent the dependencies between a number of diseases and other specific diseases. However, classical techniques systematically generate every AR meeting some minimal conditions on data frequency, hence generating a vast amount of uninteresting ARs, which need to be filtered out. The lack of manually annotated ARs has favored unsupervised filtering, even though they produce limited results. In this paper, we propose a semi-supervised system, able to identify relevant ARs among HIV-related diseases with a minimal amount of annotated training data. Our system has been able to extract a good number of relationships between HIV-related diseases that have been previously detected in the literature but are scattered and are often little known. Furthermore, a number of plausible new relationships have shown up which deserve further investigation by qualified medical experts