The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1,2,3,4,5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
State of Minnesota grant H001274506 to S.C.E. and D.F.V.; NIH GM63904 to S.C.E.; NIH grant P30DK084567 to S.C.E. and K.J.C.; NIH grant DK083219 to V.M.B.; Mayo Foundation; NIH DA032194 to K.J.C.; NIH grant R41HL108440 to D.F.C. and S.C.F.; NIH grant GM088424 to J.J.E.; NSF grant DBI0923827 to D.F.V.; General Research Fund (HKU771611, HKU771110, HKU769809M) from the Research Grant Council, The University of Hong Kong and the Tang King Yin Research Fund to A.C.M. and A.Y.H.L. We thank G. Davis for discussion on ssDNA use with custom restriction enzymes, H. J. Fadel for in vitro RNA synthesis, and S. Westcot for comments on this manuscript. We thank G. Moulder for help in DNA analyses and members of the Mayo Clinic Zebrafish Core Facility for excellent animal care.
This movie shows red blood cell movement through the blood vessels in wild type embryos.
This movie shows red blood cell movement through the blood vessels in cdh5 MO-injected embryos.
This movie shows red blood cell movement through the blood vessels in cdh5 GoldyTALEN-injected embryos.