Abstract
Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.
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Acknowledgments
This work was supported by grants from the National Institutes of Health (R01MH078972 to X.Z, U54HD090256 to the Waisman Center), John Merck Fund (to X.Z and AB), Jenni and Kyle Professorship (to X.Z), and a Rath Graduate Fellowship to JH.
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Li, M., Hunt, J.F.V.S., Bhattacharyya, A., Zhao, X. (2019). One-Step Generation of Seamless Luciferase Gene Knockin Using CRISPR/Cas9 Genome Editing in Human Pluripotent Stem Cells. In: Ben-Yosef, D., Mayshar, Y. (eds) Fragile-X Syndrome. Methods in Molecular Biology, vol 1942. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9080-1_5
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DOI: https://doi.org/10.1007/978-1-4939-9080-1_5
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