Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

By T. Čermák, Erin L. Doyle, Michelle L. Christian, Li Wang, Yong Zhang, Clarice L. Schmidt, J. Baller, N. Somia, Adam J. Bogdanove, Daniel F. Voytas. Nucleic Acids Research, 2011

2,127 citations recorded by the source corpus.

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

Subfields: Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/0d80050b042506bad4e916d74f3535df7f6ec63d

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Citations in the corpus

TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction (2012, methodology): “Users may select from four common TALEN architectures (7,13,15,24) with pre-selected ranges for spacer size and numbers of repeats or enter their own ranges for these parameters.”
Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement (2017, methodology): “TALENs belong to the second generation of genome editing tools and were first used for plant genome editing in 2011 (Cermak et al., 2011; Mahfouz et al., 2011).”
Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair (2014, background): “While sequencespecific TALE nucleases can be readily assembled from TALE repeats specific to each nucleotide (Cermak et al. 2011; Huang et al. 2011; Sander et al. 2011), sgRNAs for the CRISPR/Cas9 system can be easily generated by cloning of target-specific oligonucleotides into sgRNA expression vec”
Response of plants to water stress (2014, background): “…restriction endonucleases, such as zinc finger nucleases (ZFN) or TAL-effector nucleases (TALEN; Shukla et al., 2009; Osakabe et al., 2010b; Zhang et al., 2010; Cermak et al., 2011), and more recently, the CRISPR/CAS system (Li et al., 2013; Nekrasov et al., 2013; Shan et al., 2013).”
Genome Editing of Plants (2017, background): “These motifs have proven to be highly modular, and most engineered TAL effector arrays recognize their targets with high efficiency (Cermak et al., 2011).”
Genome Engineering of Crops with Designer Nucleases (2012, methodology): “…platforms for constructing TALENs or dTALEs. h ey include Golden Gate assembly methods that allow seamless construction of TALE repeat arrays (Cermak et al., 2011; Engler et al., 2009; Sanjana et al., 2012; Zhang et al., 2011) as well as ligation-based systems (Reyon et al., 2012; Sander et…”