Abstract
The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions.
Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.
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Benetó, N., Grinberg, D., Vilageliu, L., Canals, I. (2021). Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling. In: Turksen, K. (eds) Induced Pluripotent Stem Cells and Human Disease. Methods in Molecular Biology, vol 2549. Humana, New York, NY. https://doi.org/10.1007/7651_2021_374
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DOI: https://doi.org/10.1007/7651_2021_374
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2584-2
Online ISBN: 978-1-0716-2585-9
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