Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases

Abstract

We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg.

How to make a teleost adenohypophysis: Molecular pathways of pituitary development in zebrafish

“…At the same time, genomic tools have enormously improved, facilitating the cloning of randomly induced mutations (Dahm and Geisler, 2006, Sivasubbu et al, 2007. In addition, methods of reverse genetics have been established, including gene knock-down via the injection of specific antisense morpholino oligos (MOs) (Nasevicius and Ekker, 2000, Doyon et al, 2008, Meng et al, 2008, Wienholds et al, 2003, target-selected ENU mutagenesis (also named TILLING; targeted induced local lesions in genomes) (Nasevicius and Ekker, 2000, Doyon et al, 2008, Meng et al, 2008, Wienholds et al, 2003, and, most recently, targeted introduction of small deletions via engineered zincfinger-nucleases (Nasevicius and Ekker, 2000, Doyon et al, 2008, Meng et al, 2008, Wienholds et al, 2003.…”

Quantitative measurements of the optokinetic response in adult fish

“…The need for such assays have been low, since there are very few adult mutant strains of potential interest for vision research available. This situation is about to change with the advent of generating mutant strains with defined genetic lesions, either by TILLING (Targeting Induced Local Lesions IN Genomes) (Moens et al, 2008;Wienholds et al, 2003) or zinc finger nucleases (Doyon et al, 2008;Meng et al, 2008) and the growth of transgenic technology. Hence there will be an increasing need for robust visual assays of adult vision.…”

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

“…The advent of genomic-targeted modification technology mediated by ZFN, TALENs, and CRISPR/Cas9 gives hope to solving this technical problem. Cells or embryos of the mouse, zebrafish, other model organisms, and certain economic species have had ZFN and TALENs successfully applied to achieve site-directed mutagenesis of endogenous genes (Doyon et al 2008;Kim et al 1996;Osiak et al 2011). The CRISPR/Cas9 system has a wide range of potential applications in gene editing at the cellular level and the construction and screening of large-scale mutant libraries due to the characteristic short palindromic repeat sequences (Cermak et al 2011;Li et al 2011).…”

“…Targeted genome modification technology can be mediated by zinc finger nucleases (ZFNs) (Doyon et al 2008;Kim et al 1996;Osiak et al 2011), transcription activator-like effector nucleases (TALENs) (Bogdanove and Voytas 2011;Huang et al 2011;Sander et al 2011), and type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) (Cong et al 2013;Hwang et al 2013). TALENs are artificial nucleases that consist of DNA binding domains of transcription activator-like (TAL) effector and catalytic Fok I domains (Bogdanove and Voytas 2011).…”