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Ribonucleoproteins Mediated Efficient In Vivo Gene Editing in Skin Stem Cells

  • Wenbo Wu
  • Ting ChenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1879)

Abstract

The clustered regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system functions like an adaptive immune system in a variety of microbes and has recently been engineered as a powerful tool for manipulating genomic sequences in a huge variety of cell types. In mammals, CRISPR/Cas9 has the potential to bring curative therapies to patients with genetic diseases, although it remained unknown whether suitable in vivo methods for its use are feasible. It is now appreciated that the efficient delivery of these genome-editing tools into most tissue types, including skin, remains a major challenge. Here, we describe a detailed protocol for performing in vivo gene editing of genomic sequences in mouse skin stem cells using Cas9/sgRNAs ribonucleoproteins in combination with electrotransfer technology. We here present all of the required methods needed for the protocol, including molecular cloning, in vitro sgRNA expression and sgRNA purification, Cas9 protein purification, and in vivo delivery of cas9 ribonucleoproteins. This protocol provides a novel in vivo gene editing strategy using ribonucleoproteins for skin stem cells and can potentially be used as curative treatment for genetic diseases in skin and other somatic tissues.

Keywords

Cas9/sgRNA ribonucleoproteins Electrotransfer In vivo gene editing Skin stem cells 

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Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life SciencesPeking UniversityBeijingChina
  2. 2.National Institute of Biological SciencesBeijingChina

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