Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro

Methods Mol Biol. 2017:1521:55-68. doi: 10.1007/978-1-4939-6588-5_4.

Abstract

Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

Keywords: CRISPR/Cas9; Genome engineering; Homology-directed repair; PiggyBac transposon system; TALEN.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • CRISPR-Cas Systems
  • Cell Separation
  • Clone Cells
  • Endonucleases / metabolism*
  • Gene Editing / methods*
  • Genetic Engineering / methods*
  • Genetic Vectors / metabolism
  • HEK293 Cells
  • Homologous Recombination
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism*
  • Polymerase Chain Reaction
  • Reproducibility of Results
  • Transcription Activator-Like Effector Nucleases
  • Transfection

Substances

  • Endonucleases
  • Transcription Activator-Like Effector Nucleases