Synthetic transcriptional repressors design based on the CRISPR-Cas technology in 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 benthamian...
| Autor: | |
|---|---|
| Tipo de recurso: | tesis de maestría |
| Fecha de publicación: | 2020 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | inglés |
| OAI Identifier: | oai:riunet.upv.es:10251/136735 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/136735 |
| Access Level: | acceso abierto |
| Palabra clave: | CRISPR-Cas9 CRISPR-Cas12a N. benthamiana Represión Regulación Transcripción dCas9 dCas12a SRDX BRD KRAB SunTag BIOQUIMICA Y BIOLOGIA MOLECULAR Máster Universitario en Biotecnología Molecular y Celular de Plantas-Màster Universitari en Biotecnologia Molecular i Cel·lular de Plantes |
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oai:riunet.upv.es:10251/136735 |
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ES |
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España |
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|
| dc.title.none.fl_str_mv |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana Diseño de represores transcripcionales sintéticos basados en CRISPR-Cas en N. benthamiana. |
| title |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana |
| spellingShingle |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana Salazar-Sarasúa, Blanca CRISPR-Cas9 CRISPR-Cas12a N. benthamiana Represión Regulación Transcripción dCas9 dCas12a SRDX BRD KRAB SunTag BIOQUIMICA Y BIOLOGIA MOLECULAR Máster Universitario en Biotecnología Molecular y Celular de Plantas-Màster Universitari en Biotecnologia Molecular i Cel·lular de Plantes |
| title_short |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana |
| title_full |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana |
| title_fullStr |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana |
| title_full_unstemmed |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana |
| title_sort |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamiana |
| dc.creator.none.fl_str_mv |
Salazar-Sarasúa, Blanca |
| author |
Salazar-Sarasúa, Blanca |
| author_facet |
Salazar-Sarasúa, Blanca |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Gadea Vacas, José Orzáez Calatayud, Diego Vicente Selma García, Sara Instituto Universitario Mixto de Biología Molecular y Celular de Plantas Departamento de Biotecnología Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural Repositorio Institucional de la Universitat Politècnica de València Riunet |
| dc.subject.none.fl_str_mv |
CRISPR-Cas9 CRISPR-Cas12a N. benthamiana Represión Regulación Transcripción dCas9 dCas12a SRDX BRD KRAB SunTag BIOQUIMICA Y BIOLOGIA MOLECULAR Máster Universitario en Biotecnología Molecular y Celular de Plantas-Màster Universitari en Biotecnologia Molecular i Cel·lular de Plantes |
| topic |
CRISPR-Cas9 CRISPR-Cas12a N. benthamiana Represión Regulación Transcripción dCas9 dCas12a SRDX BRD KRAB SunTag BIOQUIMICA Y BIOLOGIA MOLECULAR Máster Universitario en Biotecnología Molecular y Celular de Plantas-Màster Universitari en Biotecnologia Molecular i Cel·lular de Plantes |
| description |
[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. |
| publishDate |
2020 |
| dc.date.none.fl_str_mv |
2020 2020-02-12 2020 2020-01-28 |
| dc.type.none.fl_str_mv |
master thesis http://purl.org/coar/resource_type/c_bdcc |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/masterThesis |
| format |
masterThesis |
| dc.identifier.none.fl_str_mv |
https://riunet.upv.es/handle/10251/136735 |
| url |
https://riunet.upv.es/handle/10251/136735 |
| dc.language.none.fl_str_mv |
Inglés eng |
| language_invalid_str_mv |
Inglés |
| language |
eng |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Reserva de todos los derechos http://rightsstatements.org/vocab/InC/1.0/ |
| dc.rights.openaire.fl_str_mv |
info:eu-repo/semantics/openAccess |
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open access http://purl.org/coar/access_right/c_abf2 Reserva de todos los derechos http://rightsstatements.org/vocab/InC/1.0/ |
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openAccess |
| dc.format.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Universitat Politècnica de València |
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Universitat Politècnica de València |
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reponame:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia instname:Universitat Politècnica de València (UPV) |
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Universitat Politècnica de València (UPV) |
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RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
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RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
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1869420770955886592 |
| spelling |
Synthetic transcriptional repressors design based on the CRISPR-Cas technology in N. benthamianaDiseño de represores transcripcionales sintéticos basados en CRISPR-Cas en N. benthamiana.Salazar-Sarasúa, BlancaCRISPR-Cas9CRISPR-Cas12aN. benthamianaRepresiónRegulaciónTranscripcióndCas9dCas12aSRDXBRDKRABSunTagBIOQUIMICA Y BIOLOGIA MOLECULARMáster Universitario en Biotecnología Molecular y Celular de Plantas-Màster Universitari en Biotecnologia Molecular i Cel·lular de Plantes[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.Universitat Politècnica de ValènciaGadea Vacas, JoséOrzáez Calatayud, Diego VicenteSelma García, SaraInstituto Universitario Mixto de Biología Molecular y Celular de PlantasDepartamento de BiotecnologíaEscuela Técnica Superior de Ingeniería Agronómica y del Medio NaturalRepositorio Institucional de la Universitat Politècnica de València Riunet20202020-02-1220202020-01-28master thesishttp://purl.org/coar/resource_type/c_bdccinfo:eu-repo/semantics/masterThesisapplication/pdfhttps://riunet.upv.es/handle/10251/136735reponame:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valénciainstname:Universitat Politècnica de València (UPV)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Reserva de todos los derechoshttp://rightsstatements.org/vocab/InC/1.0/info:eu-repo/semantics/openAccessoai:riunet.upv.es:10251/1367352026-06-13T07:49:27Z |
| score |
15,300719 |