Abstract
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.
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Acknowledgments
This work was supported by the associations Aviesan-Unadev, France Choroideremia, and Vaincre Usher 2. We thank the MRI imaging facility for FACS analysis.
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Sanjurjo-Soriano, C., Erkilic, N., Mamaeva, D., Kalatzis, V. (2021). CRISPR/Cas9-Mediated Genome Editing to Generate Clonal iPSC Lines. In: Nagy, A., Turksen, K. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 2454. Humana, New York, NY. https://doi.org/10.1007/7651_2021_362
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DOI: https://doi.org/10.1007/7651_2021_362
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