Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells
Entity
UAM. Departamento de Biología MolecularPublisher
Springer NatureDate
2022-01-01Citation
10.1007/978-1-0716-2010-6_10
Arístides López-Márquez, Ainhoa Martínez-Pizarro, Belén Pérez, Eva Richard, and Lourdes R. Desviat (2022). Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells. In: Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells V. Arechavala-Gomeza y A. Garanto (Eds). Methods in Molecular Biology (pp. 167-284). Springer Nature
ISSN
1064-3745ISBN
9781071620090DOI
10.1007/978-1-0716-2010-6_10Funded by
This book is based upon work from COST Action DARTER (CA 17103), supported by COST (European Cooperation in Science and Technology).Subjects
Splicing; Gene editing; CRISPR/Cas9; HepG2; Inherited metabolic diseases; Phenylke tonuria; Cellular models; Biología y Biomedicina / BiologíaRights
©The Editor(s) (if applicable) and The Author(s) 2022Abstract
The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based 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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Google Scholar:López-Márquez, Arístides
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Martínez-Pizarro, Ainhoa
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Pérez, Belén
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Richard Rodríguez, Eva María
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Ruiz Desviat, Lourdes
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