Diseño de represores transcripcionales sintéticos basados en CRISPR-Cas en N. benthamiana.

[EN] Deactivated versions of Cas proteins like dCas9 and dCas12a open new posibilities for plant synthetic biology in the realm of negative transcriptional regulation. Here we describe two repression strategies tested on a luciferase reporter gene through transient expression in Nicotiana benthamiana. The first one consists of a single active repression domain (BRD, SRDX and KRAB repression domains were used) fused to dCas9 or dCas12a and guided to different positions inside the promoter or the target gene. Positions -35, +51 and +62 from the TSS were selected for dCas9 and positions - 165, -66 and -9 were selected for dCas12a as the best working guides. These were then tested on pairs, increasing repression efficiency. KRAB domain was discarded from further assays for lower performance from the other domains. The second strategy tested consisted on the use of a repetitive peptide array called SunTag with the ability to recruit numerous antibody fusions. The SunTag was fused to dCas9 and dCas12a, and its antibody (ScFv) was fused to SRDX and BRD repression domains. Two SunTags were tested, one with 5 amino acids and another one with 22 amino acids as spacers between epitopes. Assays were carried out using previously selected guides alone and in pairs. Results show that dCas12a is a better endonuclease for transcriptional repression than dCas9. Both SRDX and BRD domains work, although SRDX is better for most strategies. Using more than one guide increases repression. SunTag 5aa does not seem to be able to increase repression efficiency, but recruiting more repression domains through the use of SunTag 22aa efficiently enhances repression. All in all, the best strategy out of all of the ones tested seems to be the use of dCas12a fused to the SunTag 22aa with either BRD or SRDX domains.[ES] Las nuevas tecnologías basadas en las nucleasas específicas de secuencia CRISPR-Cas han revolucionado los campos de la biología sintética y la ingeniería metabólica al permitir el desarrollo de herramientas eficientes y precisas. El sistema CRISPR-Cas se basa en la acción de una nucleasa tipo Cas a la que se asocia un pequeño guía de ARN que portará la secuencia complementaria a la diana de ADN a la que queremos dirigir la proteína. La especificidad que ofrece este sistema ha hecho que su uso esté ampliamente extendido, con un gran número de aplicaciones. En el campo de la biotecnología vegetal se ha utilizado de forma eficaz para la edición de genes, generación de knock outs o para la creación de reguladores transcripcionales sintéticos. El uso de sistemas CRISPR-Cas como regulador transcripcional sintético se ha conseguido gracias al uso de una proteína Cas9 y Cas12a catalíticamente inactiva (dCas9, dCas12a). Mediante el uso de ARN guías es posible el dirigir a la proteína dCas9 o dCas12a a la región promotora de un gen seleccionado y regular su expresión. Trabajos previos en el grupo han conseguido obtener una activación transcripcional eficiente de un gen o un grupo de genes en N. benthamiana mediante variaciones y estrategias de fusión de distintos activadores transcripcionales a dCas9 o su guía de ARN. Sin embargo, la represión transcripcional supone un reto y es necesaria para la futura obtención de circuitos de regulación transcripcional eficientes. El trabajo realizado consistirá en la puesta a punto de la represión transcripcional en N. benthamiana mediante el uso de CRISPR-Cas9 y CRISPR-Cas12a. Para ello se abordarán distintas estrategias de fusión de dominios de represión a dCas9 y dCas12a, así como la incorporación de modificaciones tanto en la estructura de la proteína como de su guía de ARN para favorecer el reclutamiento de más represores de la transcripción mediante el empleo del péptido SunTag. Los ensayos se realizarán mediante expresión transitoria por agroinfiltración en N. benthammiana sobre un gen reportero de luciferasa.Salazar Sarasúa, B. (2020). Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana. Universitat Politècnica de València. http://hdl.handle.net/10251/136735TFG

Isolation and Functional Analysis of a PISTILLATA-like MADS-Box Gene from Argan Tree (Argania spinosa)

[EN] Argan trees (Argania spinosa) belong to a species native to southwestern Morocco, playing an important role in the environment and local economy. Argan oil extracted from kernels has a unique composition and properties. Argan trees were introduced in Tunisia, where hundreds of trees can be found nowadays. In this study, we examined reproductive development in Argan trees from four sites in Tunisia and carried out the functional characterization of a floral homeotic gene in this non-model species. Despite the importance of reproductive development, nothing is known about the genetic network controlling flower development in Argania spinosa. Results obtained in several plant species established that floral organ development is mostly controlled by MADS-box genes and, in particular, APETALA3 (AP3) and PISTILLATA (PI) homologs are required for proper petal and stamen identity. Here, we describe the isolation and functional characterization of a MADS-box gene from Argania spinosa. Phylogenetic analyses showed strong homology with PI-like proteins, and the expression of the gene was found to be restricted to the second and third whorls. Functional homology with Arabidopsis PI was demonstrated by the ability of AsPI to confer petal and stamen identity when overexpressed in a pi-1 mutant background. The identification and characterization of this gene support the strong conservation of PI homologs among distant angiosperm plants.This research was supported by the Tunisian Ministry of Higher Education, Scientific Research and by a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094280-B-100).Louati, M.; Salazar-Sarasúa, B.; Roque Mesa, EM.; Beltran Porter, JP.; Salhi Hannachi, A.; Gómez Mena, MC. (2021). Isolation and Functional Analysis of a PISTILLATA-like MADS-Box Gene from Argan Tree (Argania spinosa). Plants. 10(8):1-14. https://doi.org/10.3390/plants10081665S11410