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Introducing Point Mutations into Human Pluripotent Stem Cells using Seamless Genome Editing

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  • Wang et al 2020 R1 JoVE

    Accepted author manuscript, 61.8 KB, Word document

    Licence: Creative Commons: Attribution Non-Commercial (CC-BY-NC)

Original languageEnglish
JournalJournal of Visualized Experiments (JoVE)
DOIs
Publication statusPublished - 10 May 2020

Abstract

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4α locus.

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