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Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60–70%). Currently, there are no specific cures available for CdLS and clinical management is ne...

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Autores principales: Umbach, Alessandro, Maule, Giulia, Kheir, Eyemen, Cutarelli, Alessandro, Foglia, Marika, Guarrera, Luca, Fava, Luca L., Conti, Luciano, Garattini, Enrico, Terao, Mineko, Cereseto, Anna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9438151/
https://www.ncbi.nlm.nih.gov/pubmed/36056433
http://dx.doi.org/10.1186/s13287-022-03135-0
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author Umbach, Alessandro
Maule, Giulia
Kheir, Eyemen
Cutarelli, Alessandro
Foglia, Marika
Guarrera, Luca
Fava, Luca L.
Conti, Luciano
Garattini, Enrico
Terao, Mineko
Cereseto, Anna
author_facet Umbach, Alessandro
Maule, Giulia
Kheir, Eyemen
Cutarelli, Alessandro
Foglia, Marika
Guarrera, Luca
Fava, Luca L.
Conti, Luciano
Garattini, Enrico
Terao, Mineko
Cereseto, Anna
author_sort Umbach, Alessandro
collection PubMed
description BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60–70%). Currently, there are no specific cures available for CdLS and clinical management is needed for life. Disease models are highly needed to find a cure. Among therapeutic possibilities are genome editing strategies based on CRISPR-Cas technology. METHODS: A comparative analysis was performed to test the most recent CRISPR-Cas technologies comprising base- and prime-editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed repair (HDR) induced by Cas9 nuclease activity. The HDR method that was found more efficient was applied to repair a CdLS-causing mutation in the NIPBL gene. Human-induced pluripotent stem cells (hiPSCs) derived from a CdLS patient carrying the c.5483G > A mutation in the NIPBL were modified through HDR to generate isogenic corrected clones. RESULTS: This study reports an efficient method to repair the NIPBL gene through HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed the generation of isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency. CONCLUSIONS: CdLS cellular models were generated which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular, the hiPSC-based cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that were generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-022-03135-0.
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spelling pubmed-94381512022-09-03 Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology Umbach, Alessandro Maule, Giulia Kheir, Eyemen Cutarelli, Alessandro Foglia, Marika Guarrera, Luca Fava, Luca L. Conti, Luciano Garattini, Enrico Terao, Mineko Cereseto, Anna Stem Cell Res Ther Research BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare multisystem genetic disorder which is caused by genetic defects involving the Nipped-B-like protein (NIPBL) gene in the majority of clinical cases (60–70%). Currently, there are no specific cures available for CdLS and clinical management is needed for life. Disease models are highly needed to find a cure. Among therapeutic possibilities are genome editing strategies based on CRISPR-Cas technology. METHODS: A comparative analysis was performed to test the most recent CRISPR-Cas technologies comprising base- and prime-editors which introduce modifications without DNA cleavages and compared with sequence substitution approaches through homology directed repair (HDR) induced by Cas9 nuclease activity. The HDR method that was found more efficient was applied to repair a CdLS-causing mutation in the NIPBL gene. Human-induced pluripotent stem cells (hiPSCs) derived from a CdLS patient carrying the c.5483G > A mutation in the NIPBL were modified through HDR to generate isogenic corrected clones. RESULTS: This study reports an efficient method to repair the NIPBL gene through HDR mediated by CRISPR-Cas and induced with a compound (NU7441) inhibiting non-homologous end joining (NHEJ) repair. This sequence repair method allowed the generation of isogenic wild-type hiPSCs clones with regular karyotype and preserved pluripotency. CONCLUSIONS: CdLS cellular models were generated which will facilitate the investigation of the disease molecular determinants and the identification of therapeutic targets. In particular, the hiPSC-based cellular models offer the paramount advantage to study the tissue differentiation stages which are altered in the CdLS clinical development. Importantly, the hiPSCs that were generated are isogenic thus providing the most controlled experimental set up between wild-type and mutated conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-022-03135-0. BioMed Central 2022-09-02 /pmc/articles/PMC9438151/ /pubmed/36056433 http://dx.doi.org/10.1186/s13287-022-03135-0 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Umbach, Alessandro
Maule, Giulia
Kheir, Eyemen
Cutarelli, Alessandro
Foglia, Marika
Guarrera, Luca
Fava, Luca L.
Conti, Luciano
Garattini, Enrico
Terao, Mineko
Cereseto, Anna
Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology
title Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology
title_full Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology
title_fullStr Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology
title_full_unstemmed Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology
title_short Generation of corrected hiPSC clones from a Cornelia de Lange Syndrome (CdLS) patient through CRISPR-Cas-based technology
title_sort generation of corrected hipsc clones from a cornelia de lange syndrome (cdls) patient through crispr-cas-based technology
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9438151/
https://www.ncbi.nlm.nih.gov/pubmed/36056433
http://dx.doi.org/10.1186/s13287-022-03135-0
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