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Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity

Nature Methods volume 11, pages 429–435 (2014)Cite this article

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Abstract

Although transcription activator–like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

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Figure 1: TALEN architecture and selection scheme.
Figure 2: In vitro selection results.
Figure 3: In vitro specificity as a function of TALEN length.
Figure 4: In vitro selection specificity and discrete cleavage efficiencies of TALENs containing canonical or engineered C-terminal domains.
Figure 5: Specificity of engineered TALENs in human cells.

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Acknowledgements

J.P.G., V.P. and D.R.L. were supported by Defense Advanced Research Projects Agency HR0011-11-2-0003 and N66001-12-C-4207, US National Institutes of Health (NIH) NIGMS R01 GM095501 (D.R.L.), and the Howard Hughes Medical Institute (HHMI). D.R.L. was supported as a HHMI Investigator. V.P. was supported by award T32GM007753 from US National Institute of General Medical Sciences. D.R., S.Q.T., J.D.S. and J.K.J. were supported by a NIH Director Pioneer Award (DP1 GM105378). J.K.J. was supported by the Jim and Ann Orr Massachusetts General Hospital Research Scholar Award. We thank M.L. Maeder for preforming transfections and isolating genomic DNA, and C. Khayter and M. Goodwin for technical assistance.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    John P Guilinger, Vikram Pattanayak & David R Liu

  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    John P Guilinger, Vikram Pattanayak & David R Liu

  3. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Deepak Reyon, Shengdar Q Tsai, Jeffry D Sander & J Keith Joung

  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Deepak Reyon, Shengdar Q Tsai, Jeffry D Sander & J Keith Joung

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Contributions

J.P.G., V.P., D.R., J.D.S. and S.Q.T. performed the experiments, designed the research, analyzed the data and wrote the manuscript. J.K.J. and D.R.L. designed the research, analyzed the data and wrote the manuscript.

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Correspondence to David R Liu.

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Competing interests

D.R.L. and J.K.J. have filed a provisional patent (US 61/868,846) related to this work and are consultants for Editas Medicine, a company that applies genome engineering technologies. J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

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Guilinger, J., Pattanayak, V., Reyon, D. et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 11, 429–435 (2014). https://doi.org/10.1038/nmeth.2845

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