One-Step Generation of Seamless Luciferase Gene Knockin Using CRISPR/Cas9 Genome Editing in Human Pluripotent Stem Cells
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Updated: Jan 27, 2026

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
Published on: May 10, 2020
Meng Li1, Jack Faro Vander Stoep Hunt1, Anita Bhattacharyya2,3
1Waisman Center, University of Wisconsin-Madison, Madison, WI, USA.
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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