Complete decoding of TAL effectors for DNA recognition

Yang, Junjiao; Zhang, Yuan; Yuan, Peng; Zhou, Yan; Cai, Changzu; Ren, Qingpeng; Wen, Dingqiao; Chu, Coco; Qi, Hai; Wei, Wensheng

doi:10.1038/cr.2014.19

Cited by 70 publications

(62 citation statements)

References 11 publications

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“…Matrix positions are arranged in 5′ to 3′ order with corresponding RVDs listed above (14 total Articles that might confound analysis. Accordingly, we limited the length and affinity of the host TALE (ten repeats) and tested new RVDs individually given previous reports of unexpectedly poor binding of TALEs bearing multiple tandem identical repeats 4,29 . We also avoided flanking repeats that appeared likely to interact with the queried RVD as determined by our analysis of neighbor effects (Supplementary Fig.…”

Section: Results Reliability Of the Natural Tale Codementioning

confidence: 99%

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Improved specificity of TALE-based genome editing using an expanded RVD repertoire

et al. 2015

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Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

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Section: Results Reliability Of the Natural Tale Codementioning

confidence: 99%

“…For complete study results, see supplementary Figure 9. identified as design alternatives in prior RVD studies 4,19,29 . These new RVDs could provide useful alternatives for base recognition in contexts where canonical RVDs are insufficiently specific.…”

Section: Discussionmentioning

confidence: 99%

Improved specificity of TALE-based genome editing using an expanded RVD repertoire

et al. 2015

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“…A TALE contains a series of repeat variable domains (RVDs), each targeting a single DNA nucleotide. The RVD code for all DNA nucleotides has been well characterized (62). Thus, a TALE can easily be programmed by creating an array of RVDs that bind to the nucleotides in the target DNA.…”

Section: Crispra Versus Previous Activation Methodsmentioning

confidence: 99%

The New State of the Art: Cas9 for Gene Activation and Repression

Russa

2015

Molecular and Cellular Biology

203

150

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CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies.

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“…To further determine the role of DKK1 in PA-mediated toxicity, DKK1 gene knockout clones were obtained in both HEK293T and M2182 cells using the TALEN technique [36,39], which enables the introduction of double strand DNA breaks at specific chromosomal sites. Analysis of the sequence of the TALEN-targeted genome region confirmed that the selected HEK293T/DKK1…”

Section: Effect Of Dkk1 Knockout On Pa-mediated Toxicitymentioning

confidence: 99%

Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake

Qian

Cai

Yuan

et al. 2014

Sci. China Life Sci.

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LRP6, a co-receptor for the morphogen Wnt, aids endocytosis of anthrax complexes. Here we report that Dickkopf1 (DKK1) protein, a secreted LRP6 ligand and antagonist, is also a modulator of anthrax toxin sensitivity. shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins. However, unlike the solely inhibitory effect on Wnt signaling, the effects of DKK1 overexpression on anthrax toxicity were bidirectional, depending on its endogenous expression and cell context. Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors, an event mediated by DKK1-LRP6-Kremen2 complex. Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin (LT). Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins. DKK1, anthrax toxin, LRP6, TALENs, internalization, Kremen2, receptor, Wnt Citation:Qian LL, Cai CZ, Yuan PF, Jeong SY, Yang XZ, DeAlmeida V, Ernst J, Costa M, Cohen SN, Wei WS. Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake.

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Complete decoding of TAL effectors for DNA recognition

Cited by 70 publications

References 11 publications

Improved specificity of TALE-based genome editing using an expanded RVD repertoire

Improved specificity of TALE-based genome editing using an expanded RVD repertoire

The New State of the Art: Cas9 for Gene Activation and Repression

Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake

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