Abstract
Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.
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Acknowledgements
We thank T. Cathomen for technical advice with the T7EI assay and M. Hölzel for helpful discussion. V.H. is member of the excellence cluster ImmunoSensation and supported by grants from the German Research Foundation (SFB704 and SFB670) and the European Research Council (ERC-2009-StG 243046).
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J.L.S.-B., T.S. and V.H. developed the methodology, designed experiments, analyzed the data and wrote the manuscript. J.L.S.-B., T.S., V.K. and K.H. performed the experiments. V.H. supervised the project.
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J.L.S.-B., T.S. and V.H are inventors on a patent application dealing with LIC assembly of TALE genes.
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Supplementary Figures 1–10 and Supplementary Tables 1–5, Supplementary Methods and Supplementary Note 1 (PDF 4128 kb)
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Schmid-Burgk, J., Schmidt, T., Kaiser, V. et al. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes. Nat Biotechnol 31, 76–81 (2013). https://doi.org/10.1038/nbt.2460
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DOI: https://doi.org/10.1038/nbt.2460
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