Abstract
Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.
In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.
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Acknowledgments
We thank Mervyn Mol for technical assistance. This work was supported by a Starting Grant (STEMCARDIORISK) from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation programme [H2020 European Research Council; grant agreement #638030], and a VIDI fellowship from the Netherlands Organisation for Scientific Research [Nederlandse Organisatie voor Wetenschappelijk Onderzoek NWO; ILLUMINATE; #91715303]. Schematic and workflow figures were created with BioRender.com.
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Brandão, K.O., Grandela, C., Yiangou, L., Mummery, C.L., Davis, R.P. (2021). CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells. 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_368
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DOI: https://doi.org/10.1007/7651_2021_368
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Publisher Name: Humana, New York, NY
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