Abstract
The ability to engineer specific mutations in human embryonic stem cells (ECSs) or induced pluripotent stem cells (iPSCs) is extremely important in the modeling of human diseases and the study of biological processes. While CRISPR/Cas9 can robustly generate gene knockouts (KOs) and gene loci modifications in coding sequences of iPSCs, it remains difficult to produce monoallelic mutations or modify specific nucleotides in noncoding sequences due to technical constraints.
Here, we describe how to leverage cytosine (BE4max) and adenine (ABEmax) base editors to introduce precise mutations in iPSCs without inducing DNA double-stranded breaks. This chapter illustrates how to design and clone gRNAs, evaluate editing efficiency, and detect genomic edits at specific sites in iPSCs through the utilization of base editing technology.
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Acknowledgments
This research was supported by NIH grants U01HL134696, R01DK118155, R01DK123162, and UG3DK122644.
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Pavani, G., Klein, J.G., French, D.L., Gadue, P. (2022). Genome Engineering Human ESCs or iPSCs with Cytosine and Adenine Base Editors. In: Turksen, K. (eds) Embryonic Stem Cell Protocols . Methods in Molecular Biology, vol 2520. Humana, New York, NY. https://doi.org/10.1007/7651_2022_461
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DOI: https://doi.org/10.1007/7651_2022_461
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Publisher Name: Humana, New York, NY
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