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...
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| 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 |
| Sumario: | [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. |
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