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A suicidal zinc finger nuclease expression coupled with a surrogate reporter for efficient genome engineering

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Abstract

Genome editing with engineered nucleases, such as zinc-finger nucleases (ZFNs) and TALE nucleases, remains confronted with a high risk of cellular toxicity induced by off-targeting. Here we describe the construction of a suicidal nuclease expression vector in which a pair of ZFNs genes were flanked of its target sites. To further enrich the targeted cells, the suicidal ZFN expression cassette was also inserted within an eGFP reporter, to disrupt the ORF of eGFP gene. ZFN-associated toxicity was reduced by ~40 % with this new system, and the activities of ZFNs were ~4.5 % lower. We conclude that using this new suicidal ZFN expression and surrogate reporter system represents an improvement for genomic editing by reducing toxicity and allowing easy detection of edited cells by eGFP analysis.

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Acknowledgments

We thank Joe Mymryk for critical reading of the manuscript. This work was partially supported by Grants from National Natural Science Foundation of China (NSFC) (31172186) and 973 Program (2011CBA01002).

Supporting information

Supplementary Table 1—Primers of construction suicidal reporter vector

Author information

Authors and Affiliations

  1. College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, 712100, Shaanxi, China

    Cunfang Zhang, Kun Xu, Ling Wang, Tingting Zhang, Chonghua Ren & Zhiying Zhang

  2. Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China

    Cunfang Zhang & Linyong Hu

  3. School of Biological Science Technology and Engineering, Shaanxi University of Technology, Hanzhong, China

    Ling Wang

Authors
  1. Cunfang Zhang
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  2. Kun Xu
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  3. Linyong Hu
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  4. Ling Wang
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  5. Tingting Zhang
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  6. Chonghua Ren
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  7. Zhiying Zhang
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Corresponding author

Correspondence to Zhiying Zhang.

Additional information

Cunfang Zhang and Kun Xu have contributed equally to this work.

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Supplementary material 1 (DOC 33 kb)

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Zhang, C., Xu, K., Hu, L. et al. A suicidal zinc finger nuclease expression coupled with a surrogate reporter for efficient genome engineering. Biotechnol Lett 37, 299–305 (2015). https://doi.org/10.1007/s10529-014-1690-3

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  • DOI: https://doi.org/10.1007/s10529-014-1690-3

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