Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR

Methods Mol Biol. 2018;1768:349-362. doi: 10.1007/978-1-4939-7778-9_20.

Abstract

Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.

Keywords: CRISPR/Cas9; Genome editing; HDR; NHEJ; TALEN; ddPCR.

Publication types

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

MeSH terms

  • DNA / genetics
  • DNA / isolation & purification*
  • DNA End-Joining Repair / genetics*
  • Gene Editing / methods
  • Genetic Loci / genetics*
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells
  • Polymerase Chain Reaction / instrumentation
  • Polymerase Chain Reaction / methods*
  • Recombinational DNA Repair / genetics*

Substances

  • DNA