Detecting Single-Nucleotide Substitutions Induced by Genome Editing

Cold Spring Harb Protoc. 2016 Aug 1;2016(8). doi: 10.1101/pdb.top090845.


The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells.

MeSH terms

  • Animals
  • Gene Editing*
  • Humans
  • Nucleotides / analysis*
  • Nucleotides / genetics*
  • Oligonucleotide Probes / genetics
  • Pluripotent Stem Cells
  • Polymerase Chain Reaction / methods*


  • Nucleotides
  • Oligonucleotide Probes