title: PCR artifact in testing for homologous recombination in genomic editing in zebrafish.
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.
authors.affiliation: Section on Developmental Biology, DDB, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, United States of America.|Department of Pharmacology, College of Medicine, Chungnam National University, Jung-gu, Daejeon, Korea (ROK).
authors.forename: Minho
authors.initials: M
authors.lastname: Won
authors.affiliation: Section on Developmental Biology, DDB, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, United States of America.
authors.forename: Igor B
authors.identifier: http://orcid.org/0000-0003-0579-5693
authors.initials: IB
authors.lastname: Dawid
mesh_terms: D000818:Animals; D016477:Artifacts; D015139:Blotting, Southern; D000072669:Gene Editing; D059765:Homologous Recombination / Q000235:genetics; D016133:Polymerase Chain Reaction; D015027:Zebrafish / Q000235:genetics*