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Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus

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Abstract

The Streptococcus thermophilus CRISPR3-Cas (StCas9) system has been shown to mediate DNA cleavage in its original host and in E. coli as well as in vitro. Here, we have reconstituted the StCas9 system in yeast and conducted a systematic optimization of the sgRNA structure, including the minimal length of tracrRNA, loop structure, Match II region, Bulge motif, the minimal length of guide sequence within the crRNA, tolerance of mismatches and target sequence preference. The optimal sgRNA design for the StCas9 system achieved up to 12 and 40 % targeting efficiencies in yeast and human cells, respectively. This study provides important insight into the sequence and structural requirements necessary to develop a targeted and highly efficient eukaryotic gene editing platform using CRISPR-Cas systems.

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Acknowledgments

The authors would like to thank the colleagues in Professor Zhang’s lab for their excellent technical assistance and helpful discussions. This work was supported by National Science and Technology Major Project of China [2014ZX0801009B].

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Authors and Affiliations

  1. Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Kun Xu, Chonghua Ren, Zhongtian Liu, Tao Zhang, Tingting Zhang, Duo Li, Ling Wang, Qiang Yan, Lijun Guo, Juncen Shen & Zhiying Zhang

  2. School of Biological Science Technology and Engineering, Shaanxi University of Technology, Hanzhong, 723000, Shaanxi, China

    Tao Zhang & Ling Wang

  3. Research Institute of Applied Biology, Shanxi University, Taiyuan, 030006, Shanxi, China

    Tingting Zhang

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  1. Kun Xu
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  2. Chonghua Ren
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  3. Zhongtian Liu
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Correspondence to Zhiying Zhang.

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K. Xu and C. Ren contributed equally to this work.

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Xu, K., Ren, C., Liu, Z. et al. Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus . Cell. Mol. Life Sci. 72, 383–399 (2015). https://doi.org/10.1007/s00018-014-1679-z

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