In vivo genome editing using a high-efficiency TALEN system

Abstract

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease using 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 applications1–5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show this enhanced TALEN toolkit demonstrates 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 (MO)-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA (ssDNA) oligonucleotides (oligos) to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair (HDR), 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.

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

“…Gene targeting via non-homologous end-joining (NHEJ) has also been suggested to generate combined reporter and knockout alleles 9 . Combination of TALEN-based gene targeting with insertion of a few base pairs or transgenes using small ssDNA oligonucleotides or donor vectors with longer homologous arms was initially reported for zebrafish 10,11 and later extended to other animal models including mouse 12,13 . The mouse remains the most widely used animal model in the study of human biology and diseases.…”

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

“…More recently, the transcription activator-like effectors (TALEs) technology, a milestone in the development of zebrafish mutant and transgenic lines, has lifted the limit of loci-specific targeting. With very low off-targeting effects, TALEs were therefore the first successful genome editing method that permitted homologous-directed recombination (HDR) and NHEJ-mediated knockin in zebrafish (Bedell et al 2012;Zu et al 2013). Two reports (Chang et al 2013;Hwang et al 2013b) showed that double stranded breaks (DSB), which are simpler in design and have higher mutagenesis efficiency, could also be generated using the Crispr/Cas9 technology based on the same approach used by Bedell et al (2012).…”

“…With very low off-targeting effects, TALEs were therefore the first successful genome editing method that permitted homologous-directed recombination (HDR) and NHEJ-mediated knockin in zebrafish (Bedell et al 2012;Zu et al 2013). Two reports (Chang et al 2013;Hwang et al 2013b) showed that double stranded breaks (DSB), which are simpler in design and have higher mutagenesis efficiency, could also be generated using the Crispr/Cas9 technology based on the same approach used by Bedell et al (2012). Following these studies, Hruscha et al (2013) achieved the integration of HA-tags into the sequence of single strand oligonucleotides flanked by two short homology arms of the targeted gene.…”

Neuroscience Research Using Non-human Primate Models and Genome Editing

“…Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system are mainly used as engineered nucleases. A number of genetically modified animals have already been generated using such restriction enzymes (Bedell et al 2012;Geurts et al 2009;Hauschild et al 2011;Mashimo et al 2010;Ochiai et al 2010;Sung et al 2013;Suzuki et al 2013;Wang et al 2013;Yang et al 2013). Among these, the CRISPR/Cas system was developed the most recently and is particularly promising (Cong et al 2013;Mali et al 2013).…”