A Southern Blot Protocol to Detect Chimeric Nuclease-Mediated Gene Repair

Methods Mol Biol. 2014;1114:325-38. doi: 10.1007/978-1-62703-761-7_21.

Abstract

Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a low-efficiency process. However, the effectiveness of such so-called genome surgery or genome editing has recently been drastically improved through technical developments, chiefly the use of designer nucleases like zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas nucleases. These enzymes are custom designed to recognize long target sites and introduce double-strand breaks (DSBs) at specific target loci in the genome, which in turn mediate significant improvements in the frequency of homologous recombination. Here, we describe a Southern blot-based assay that allows detection of gene repair and estimation of repair frequencies in a cell population, useful in cases where the targeted modification itself cannot be detected by restriction digest. This is achieved through detection of a silent restriction site introduced alongside the desired mutation, in our particular example using integration-deficient lentiviral vectors (IDLVs) coding for ZFNs and a suitable DNA repair template.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Southern* / methods
  • DNA Repair*
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Fibroblasts / metabolism
  • Genetic Vectors / genetics
  • Homologous Recombination
  • Lentivirus / genetics
  • Mice
  • Transduction, Genetic
  • Zinc Fingers / physiology*

Substances

  • Endonucleases