A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish

Liu, Yun; Luo, Daji; Lei, Yong; Hu, Wei; Zhao, Hui; Chen, Cheng

doi:10.1016/j.ymeth.2014.02.011

Cited by 51 publications

(37 citation statements)

References 44 publications

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“…Unfortunately, in earlier reports, there was no loss of gene function and the expected phenotype was not detected after gene knockout (Ansai et al 2013(Ansai et al , 2014. In this present study, to show that TALENs can be used to conduct gene knockout and gene-editing studies in medaka, a pre-built modified TALEN (Liu et al 2014) system was used to knock out an exogenous transgene in medaka.…”

Section: Introductionmentioning

confidence: 76%

“…Based on the Golden Gate method (Cong et al 2013), a modified TALEN system was developed to be suitable for eukaryotic genes as it was originally only applicable to prokaryotic gene knockouts (Liu et al 2013(Liu et al , 2014. This modified TALEN platform was used to efficiently induce mutations in Xenopus and zebrafish.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…First, BsaI digested and identified A (NI), T (NG), C (HD), and G (NN) single modular plasmid, and then T4 ligase was used to link and construct the recombinant plasmids pFUS_A and pFUS_Bn. Second, pCS2-GFP-TALEN-ELD (left targeting recognition sequences plasmid) and pCS2-GFP-TALEN-KKR (right targeting recognition sequences plasmid) were constructed by digestion-connection (Liu et al 2014). The detection primers used for plasmid construction were as follows:…”

Section: Gfp-talen Plasmid Constructionmentioning

confidence: 99%

“…Once the DSB was repaired by TALEN, this would effectively cause critical changes in the amino acids in the Y66 region, blocking the translation of the GFP protein and thus preventing green fluorescence. TALEN knockout plasmids pCS2-GFP-TALEN-ELD and pCS2-GFP-TALEN-KKR were constructed by a two-step method of enzyme digestion-connection (Liu et al 2014). …”

Section: Design and Construction Of Talensmentioning

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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Section: Introductionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Gfp-talen Plasmid Constructionmentioning

confidence: 99%

Section: Design and Construction Of Talensmentioning

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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Accelerated genome engineering through multiplexing

Bao

Cobb

Zhao

2015

WIREs Mechanisms of Disease

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Throughout the biological sciences, the past fifteen years have seen a push towards the analysis and engineering of biological systems at the organism level. Given the complexity of even the simplest organisms, though, to elicit a phenotype of interest often requires genotypic manipulation of several loci. By traditional means, sequential editing of genomic targets requires a significant investment of time and labor, as the desired editing event typically occurs at a very low frequency against an overwhelming unedited background. In recent years, the development of a suite of new techniques has greatly increased editing efficiency, opening up the possibility for multiple editing events to occur in parallel. Termed as multiplexed genome engineering, this approach to genome editing has greatly expanded the scope of possible genome manipulations in diverse hosts, ranging from bacteria to human cells. The enabling technologies for multiplexed genome engineering include oligonucleotide-based and nuclease-based methodologies, and their application has led to the great breadth of successful examples described in this review. While many technical challenges remain, there also exists a multiplicity of opportunities in this rapidly expanding field.

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