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Applied Biochemistry and Biotechnology

, Volume 180, Issue 4, pp 655–667 | Cite as

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

  • Yi Yang
  • Songcai Liu
  • Yunyun Cheng
  • Linyan Nie
  • Chen Lv
  • Gang Wang
  • Yu Zhang
  • Linlin HaoEmail author
Article

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.

Keywords

Cas9/gRNA SSA reporter assay HR reporter assay T7EI 

Notes

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).

Compliance with Ethical Standards

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yi Yang
    • 1
  • Songcai Liu
    • 1
  • Yunyun Cheng
    • 1
  • Linyan Nie
    • 1
  • Chen Lv
    • 1
  • Gang Wang
    • 1
  • Yu Zhang
    • 1
  • Linlin Hao
    • 1
    Email author
  1. 1.College of Animal ScienceJilin UniversityChangchunChina

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