PCR artifact in testing for homologous recombination in genomic editing in zebrafish

PLoS One. 2017 Mar 31;12(3):e0172802. doi: 10.1371/journal.pone.0172802. eCollection 2017.

Abstract

We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.

MeSH terms

  • Animals
  • Artifacts
  • Blotting, Southern
  • Gene Editing
  • Homologous Recombination / genetics
  • Polymerase Chain Reaction
  • Zebrafish / genetics*

Grants and funding

This work was funded by the intramural research program of the Eugene Kennedy Shriver National Institute for Child Health and Human Development, under program number ZIA HD 008809. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. However, manuscripts emanating from the intramural research program are subject to a clearance procedure, and this manuscript has been cleared.