Abstract
Transcription activator–like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.
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Acknowledgements
This work was supported by the National Research Foundation of Korea (J.-S.K., 2012-0001225), the Intelligent Synthetic Biology Center of the Global Frontier Project funded by the Ministry of Education, Science and Technology, Korea (D.B., 2011-0031956), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (S.K., 311062-04-2-sb1010), and Plant Molecular Breeding Center of Next-Generation BioGreen 21 Program (S.K., PJ009081).
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J.-S.K., S.K. and D.B. supervised the research and wrote the manuscript. All the other authors performed the experiments.
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J.Y.Y., M.S.L., E.M.G., H.J.S. and S.K. are employees of ToolGen.
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Kim, Y., Kweon, J., Kim, A. et al. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31, 251–258 (2013). https://doi.org/10.1038/nbt.2517
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DOI: https://doi.org/10.1038/nbt.2517
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