Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1

Methods Mol Biol. 2018;1772:233-248. doi: 10.1007/978-1-4939-7795-6_13.

Abstract

Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

Keywords: CRISPR-Cas9; Gene editing; Gene knock out; Genome engineering.

MeSH terms

  • Cell Line, Tumor
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair / genetics
  • Endonucleases / genetics*
  • Gene Editing / methods
  • Gene Knockout Techniques / methods
  • Genetic Engineering / methods
  • Genetic Vectors / genetics*
  • Genome / genetics
  • HCT116 Cells
  • Homozygote
  • Humans
  • Jagged-1 Protein / genetics*
  • RNA, Guide / genetics
  • Sequence Deletion / genetics*

Substances

  • JAG1 protein, human
  • Jagged-1 Protein
  • RNA, Guide
  • Endonucleases