Optimizing CRISPR-Cas technologies in Caenorhabditis elegans : Nested CRISPR and expanded targeting with Cas variants
In this thesis, I present an alternative, cloning-free method for the generation of endogenous fluorescent reporters in the nematode Caenorhabditis elegans. I demonstrate that Nested CRISPR is an efficient method that can be customized for the insertion of a suite of fluorescent tags and epitopes at...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2021 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/672604 |
| Acceso en línea: | http://hdl.handle.net/10803/672604 |
| Access Level: | acceso abierto |
| Palabra clave: | Genome editing CRISPR Caenorhabditis elegnas Genetic engineering Cas9 variants Edición genómica Caernohabditis elegans Ingeniería genética Variantes de Cas9 575 |
| Sumario: | In this thesis, I present an alternative, cloning-free method for the generation of endogenous fluorescent reporters in the nematode Caenorhabditis elegans. I demonstrate that Nested CRISPR is an efficient method that can be customized for the insertion of a suite of fluorescent tags and epitopes at endogenous loci using a combination of single-stranded and double-stranded DNA repair templates. In this thesis, I also demonstrate the use of enzymes other than Cas9 to target non-NGG PAM sites. The results show that AsCas12a can perform efficient genome editing in TTTV PAMs. Furthermore, the structurally engineered Cas9 variants SpG and SpRY can mediate genome editing in NGN and NYN PAMs, respectively, via both error-prone and precise repair mechanisms under optimized conditions. |
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