1. Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells.
- Author
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Xu H, Kita Y, Bang U, Gee P, and Hotta A
- Subjects
- Adult, DNA, Single-Stranded genetics, Female, Homologous Recombination genetics, Humans, Male, RNA, Guide, CRISPR-Cas Systems genetics, RNA, Guide, CRISPR-Cas Systems metabolism, CRISPR-Cas Systems genetics, Electroporation methods, Gene Editing methods, Pluripotent Stem Cells cytology, Pluripotent Stem Cells metabolism, Ribonucleoproteins genetics, Ribonucleoproteins metabolism
- Abstract
Selection-free, scarless genome editing in human pluripotent stem cells (PSCs) by utilizing ribonucleoprotein (RNP) of CRISPR-Cas9 is a useful tool for a variety of applications. However, the process can be hampered by time-consuming subcloning steps and inefficient delivery of the RNP complex and ssDNA template. Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al. (2019)., Competing Interests: P.G. is an employee of MaxCyte Inc. The other authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
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