Targeted gene disruption in somatic zebrafish cells using engineered TALENs

Sander, Jeffry D.; Cade, Lindsay; Khayter, Cyd; Reyon, Deepak; Peterson, Randall T.; Joung, J. Keith; Yeh, Jing-Ruey Joanna

doi:10.1038/nbt.1934

Cited by 563 publications

(430 citation statements)

References 13 publications

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“…This technology has been successfully applied in several animal models including mouse, rat, zebrafish and Caenorhabditis elegans [4][5][6][7][8] . Gene targeting via non-homologous end-joining (NHEJ) has also been suggested to generate combined reporter and knockout alleles 9 .…”

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confidence: 99%

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

et al. 2014

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Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.

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confidence: 99%

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

et al. 2014

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“…This modified TALEN platform was used to efficiently induce mutations in Xenopus and zebrafish. Although previous studies have shown that TALEN has high specificity (Bogdanove and Voytas 2011;Huang et al 2011;Hwang et al 2013;Lei et al 2012;Liu et al 2013Liu et al , 2014Sander et al 2011), offtarget problems remained underestimated. On the one hand, while HD, NI, and NG RVDs of TALENs could specifically correspond with C, A, and T, only NN can identify both G and A, indicating that the more G in the target sequence, the more NN is required in the corresponding synthetic TALEN artificial nucleases, but this increases the opportunity for nonspecific knockouts.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

“…specificity is determined by the repeat variant di-residues (RVDs) at the 12th and 13th amino acid positions of the TAL. The RVDs NI, NG, HD, and NN can identify corresponding adenine (A), thymine (T), cytosine (C), and guanine (G), respectively (Cermak et al 2011;Huang et al 2011;Li et al 2011;Sander et al 2011). These DNA binding domain repeating units of the TAL effector pair with DNA to form a DNA double-stranded structure at the interval region.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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Transcription activator-like effector nucleases (TALENs) are used for gene knockout and genome-editing studies in zebrafish, and these techniques have the potential to be applied to other fish species. Here, we show that TALENs can directly knock out a green fluorescent protein (GFP) transgene in medaka by affecting translation and synthesis of the GFP. We constructed a transgenic plasmid (pGFP-RFP) carrying the GFP and red fluorescent protein (RFP) genes, and used a modified TALEN method to assemble a pair of TALENs for the core chromophore Y66 region of GFP. Embryo toxicity of TALEN messenger RNA (mRNA) was far lower than the linearized plasmid; meanwhile, 76.3 % embryos, green fluorescence of embryos decreased significantly after co-injection of TALEN mRNA and the linearized plasmid, but red fluorescence showed no significant change. Real-time quantitative polymerase chain reaction and sequencing results showed that nearly 100 % mutated GFP position was disrupted at the Y66 region of GFP in the coinjected medaka embryos, caused by TALENs. This led to random insertion-deletion of nucleotides, which affected the translation of GFP and disrupted GFP synthesis. This provides new experimental evidence for designing TALEN sites in genes for which only key functional domains are known. Our results show that a modified TALEN method can efficiently and specifically mediate a transgene knockout in medaka. This report may promote the application of TALENs in gene-editing studies of fish species other than zebrafish.

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“…The TALENs have rapidly gained prominence as a novel genome-editing tool, which were successfully applied to create site-specific gene modifications in model organisms such as yeast, plants, zebra fish, mouse, rat and human cells, including human pluripotent cells. [58][59][60][61][62] TALEN has also been used to generate single base-pair mutations, linking single-nucleotide polymorphisms to specific human disease. 63 Furthermore, TALENs have even been utilized to eliminate the mutant form of mitochondrial DNA from patient-derived cells.…”

Section: Talenmentioning

confidence: 99%

A practical guide to induced pluripotent stem cell research using patient samples

Santostefano

Hamazaki

Biel

et al. 2015

Laboratory Investigation

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Approximately 3 years ago, we assessed how patient induced pluripotent stem cell (iPSC) research could potentially impact human pathobiology studies in the future. Since then, the field has grown considerably with numerous technical developments, and the idea of modeling diseases 'in a dish' is becoming increasingly popular in biomedical research. Likely, it is even acceptable to include patient iPSCs as one of the standard research tools for disease mechanism studies, just like knockout mice. However, as the field matures, we acknowledge there remain many practical limitations and obstacles for their genuine application to understand diseases, and accept that it has not been as straightforward to model disorders as initially proposed. A major practical challenge has been efficient direction of iPSC differentiation into desired lineages and preparation of the large numbers of specific cell types required for study. Another even larger obstacle is the limited value of in vitro outcomes, which often do not closely represent disease conditions. To overcome the latter issue, many new approaches are underway, including three-dimensional organoid cultures from iPSCs, xenotransplantation of human cells to animal models and in vitro interaction of multiple cell types derived from isogenic iPSCs. Here we summarize the areas where patient iPSC studies have provided truly valuable information beyond existing skepticism, discuss the desired technologies to overcome current limitations and include practical guidance for how to utilize the resources. Undoubtedly, these human patient cells are an asset for experimental pathology studies. The future rests on how wisely we use them.

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Targeted gene disruption in somatic zebrafish cells using engineered TALENs

Cited by 563 publications

References 13 publications

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

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

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

A practical guide to induced pluripotent stem cell research using patient samples

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