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Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter

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Abstract

The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (31101781, 31072102) and the Programs Foundation of Ministry of Education of China (20110061110081).

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    Authors and Affiliations

    1. College of Animal Science, Jilin University, Changchun, 130062, China

      Yi Yang, Songcai Liu, Yunyun Cheng, Linyan Nie, Chen Lv, Gang Wang, Yu Zhang & Linlin Hao

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    1. Yi Yang
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    2. Songcai Liu
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    3. Yunyun Cheng
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    8. Linlin Hao
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    Correspondence to Linlin Hao.

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    The authors declare that they have no conflicts of interest.

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    Yi Yang and Songcai Liu contributed equally to this work.

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    Yang, Y., Liu, S., Cheng, Y. et al. Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter. Appl Biochem Biotechnol 180, 655–667 (2016). https://doi.org/10.1007/s12010-016-2122-8

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