Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

Sci Rep. 2016 Mar 31:6:23549. doi: 10.1038/srep23549.

Abstract

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Publication types

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

MeSH terms

  • Biological Assay
  • CRISPR-Cas Systems
  • Cell Line
  • DNA Breaks, Double-Stranded*
  • DNA Breaks, Single-Stranded*
  • DNA End-Joining Repair*
  • Gene Editing*
  • Genetic Loci
  • Genome, Human*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Polymerase Chain Reaction
  • Recombinational DNA Repair*
  • Transcription Activator-Like Effector Nucleases / genetics*
  • Transcription Activator-Like Effector Nucleases / metabolism
  • Transfection

Substances

  • Transcription Activator-Like Effector Nucleases