CRISPR/Cas9-Mediated Genome Editing to Generate Clonal iPSC Lines

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.)
Main Authors Sanjurjo-Soriano, Carla, Erkilic, Nejla, Mamaeva, Daria, Kalatzis, Vasiliki
Format Journal Article
LanguageEnglish
Published United States 24.03.2021
Subjects
Single-cell sorting
iPSCs
Genome editing
CRISPR/Cas9
Nucleofection
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Summary:The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian cells. The coupling of both human (h)iPSCs and CRISPR/Cas9 technology offers great promise for cell therapy and regenerative medicine. However, several limitations including time and labor consumption, efficiency and efficacy of the system, and the potential off-targets effects induced by the Cas9 nuclease still need to be addressed. Here, we describe a detailed method for easily engineering genetic changes in hiPSCs, using a nucleofection-mediated protocol to deliver the CRISPR/Cas9 components into the cells, and discuss key points to be considered when designing your experiment. The clonal, genome-edited hiPSC line generated via our method can be directly used for downstream applications.
ISSN:1940-6029
DOI:10.1007/7651_2021_362