Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.

CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes / Muto, Valentina; Benigni, Federica; Magliocca, Valentina; Borghi, Rossella; Flex, Elisabetta; Pallottini, Valentina; Rosa, Alessandro; Compagnucci, Claudia; Tartaglia, Marco. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 24:9(2023). [10.3390/ijms24097988]

CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes

Rosa, Alessandro;
2023

Abstract

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.
2023
CRISPR/Cas9 technology; PEBAT; TBCD; gene editing; iPSCs; isogenic controls; neurodevelopmental disorder
01 Pubblicazione su rivista::01a Articolo in rivista
CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes / Muto, Valentina; Benigni, Federica; Magliocca, Valentina; Borghi, Rossella; Flex, Elisabetta; Pallottini, Valentina; Rosa, Alessandro; Compagnucci, Claudia; Tartaglia, Marco. - In: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. - ISSN 1422-0067. - 24:9(2023). [10.3390/ijms24097988]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1679606
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