Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase

This protocol uses lipofectamine to deliver base editors (i.e., dCas9 and AIDx fusion protein) and sgRNA expression vectors into Duchenne Muscular Dystrophy (DMD) patient-derived human induced pluripotent stem cells (hiPSCs). This protocol details mutation of the 5′ splice site of DMD exon50 with TA...

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Detalles Bibliográficos
Autores principales: Ma, Yunqing, Yuan, Juanjuan, Chang, Xing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580208/
https://www.ncbi.nlm.nih.gov/pubmed/33111067
http://dx.doi.org/10.1016/j.xpro.2019.100005
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author Ma, Yunqing
Yuan, Juanjuan
Chang, Xing
author_facet Ma, Yunqing
Yuan, Juanjuan
Chang, Xing
author_sort Ma, Yunqing
collection PubMed
description This protocol uses lipofectamine to deliver base editors (i.e., dCas9 and AIDx fusion protein) and sgRNA expression vectors into Duchenne Muscular Dystrophy (DMD) patient-derived human induced pluripotent stem cells (hiPSCs). This protocol details mutation of the 5′ splice site of DMD exon50 with TAM (targeted AID-induced mutagenesis) followed by amplicon-based NGS library preparation for high-throughput sequencing analysis. This protocol can be generalized for base editing in other hIPSCs and for correcting aberrant splicing associated with other genetic diseases. For complete information on the generation and use of this protocol, please refer to Yuan et al. (2018).
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spelling pubmed-75802082020-10-26 Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase Ma, Yunqing Yuan, Juanjuan Chang, Xing STAR Protoc Protocol This protocol uses lipofectamine to deliver base editors (i.e., dCas9 and AIDx fusion protein) and sgRNA expression vectors into Duchenne Muscular Dystrophy (DMD) patient-derived human induced pluripotent stem cells (hiPSCs). This protocol details mutation of the 5′ splice site of DMD exon50 with TAM (targeted AID-induced mutagenesis) followed by amplicon-based NGS library preparation for high-throughput sequencing analysis. This protocol can be generalized for base editing in other hIPSCs and for correcting aberrant splicing associated with other genetic diseases. For complete information on the generation and use of this protocol, please refer to Yuan et al. (2018). Elsevier 2020-06-03 /pmc/articles/PMC7580208/ /pubmed/33111067 http://dx.doi.org/10.1016/j.xpro.2019.100005 Text en © 2019 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Ma, Yunqing
Yuan, Juanjuan
Chang, Xing
Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase
title Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase
title_full Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase
title_fullStr Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase
title_full_unstemmed Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase
title_short Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase
title_sort genetic modulation of rna splicing with a crispr-guided cytidine deaminase
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580208/
https://www.ncbi.nlm.nih.gov/pubmed/33111067
http://dx.doi.org/10.1016/j.xpro.2019.100005
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AT yuanjuanjuan geneticmodulationofrnasplicingwithacrisprguidedcytidinedeaminase
AT changxing geneticmodulationofrnasplicingwithacrisprguidedcytidinedeaminase

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