Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells
The field of splice modulating RNA therapy has gained new momentum with FDA approved antisensebased drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate...
| Autores: | , , , , |
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| Formato: | otro |
| Estado: | Versión publicada |
| Fecha de publicación: | 2022 |
| País: | España |
| Recursos: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/411449 |
| Acesso em linha: | http://hdl.handle.net/10261/411449 |
| Access Level: | acceso abierto |
| Palavra-chave: | Splicing Gene editing CRISPR/Cas9 HepG2 Inherited metabolic diseases Phenylketonuria Cellular models |
| Resumo: | The field of splice modulating RNA therapy has gained new momentum with FDA approved antisensebased drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients’ samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene. Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/ Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant, which should result in the accurate recapitulation of the splicing defect. |
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