FLASH assembly of TALENs for high-throughput genome editing

Abstract

Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.

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Figure 1: Graphical overview of the FLASH assembly method.
Figure 2: Gene-disruption activities of 48 FLASH-assembled TALENs targeted to EGFP.
Figure 3: Computationally-derived design guidelines do not show statistically significant correlation with TALEN activity levels.
Figure 4: DNA sequences and frequencies of FLASH TALEN-induced mutations at endogenous human genes.

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Acknowledgements

We thank T. Cathomen for providing polyclonal U2OS-EGFP reporter cells and a T7EI protocol, Y. Fu for deriving the clonal U2OS-EGFP cell line, D. Dobbs for support and encouragement, and M. Maeder, J. Angstman and C. Ramirez for helpful comments. This work was supported by a US National Institutes of Health (NIH) Director's Pioneer Award DP1 OD006862 (J.K.J.), NIH P50HG005550 (J.K.J.), the Jim and Ann Orr MGH Research Scholar Award (J.K.J.), NIH T32 CA009216 (J.D.S.) and National Science Foundation DBI-0923827 (D.R.).

Author information

J.D.S. and J.K.J. conceived of the FLASH method. D.R., S.Q.T., C.K., J.A.F. and J.D.S. performed experiments. D.R., S.Q.T., C.K., J.A.F., J.D.S. and J.K.J. wrote the manuscript.

Correspondence to Jeffry D Sander or J Keith Joung.

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Competing interests

J.D.S. and J.K.J. are inventors on a patent application describing the FLASH method. J.K.J. is a member of the Scientific Advisory Board of Transposagen Biopharmaceuticals, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Discussion, Supplementary Tables and Supplementary Figures 1–7 (PDF 6093 kb)

Supplementary Table 1

DNA sequences encoding individual TALE repeats used to construct the archive of preassembled units required for FLASH assembly (XLSX 9 kb)

Supplementary Table 2

Archive of 376 plasmids encoding pre-assembled TALE repeat units required to practice FLASH assembly (XLSX 48 kb)

Supplementary Table 3

EGFP reporter gene sequences targeted by 48 pairs of FLASH TALENs (XLSX 15 kb)

Supplementary Table 4

Endogenous human gene sequences targeted by 96 pairs of FLASH TALENs (XLSX 17 kb)

Supplementary Table 5

PCR primers and conditions used to amplify TALEN-targeted sequences of 96 endogenous human genes (XLSX 16 kb)

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Reyon, D., Tsai, S., Khayter, C. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30, 460–465 (2012). https://doi.org/10.1038/nbt.2170

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