4.4 Article

Development of a CRISPR-Cas9 Based Luciferase Turn-On System as Nonhomologous End Joining Pathway Reporter

期刊

CHEMBIOCHEM
卷 22, 期 12, 页码 2177-2181

出版社

WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202100128

关键词

CRISPR-Cas9; DNA damage; inhibitors; non-homologous end joining (NHEJ); luciferase

资金

  1. National Natural Science Foundation of China [31870860, 81772840, 31971388]
  2. State Key Laboratory of Medicinal Chemical Biology (Nankai University) [2018103]

向作者/读者索取更多资源

A CRISPR-Cas9 based luciferase turn-on system was developed as an NHEJ pathway reporter, with nucleotide substitution creating a silent bioluminescent signal in reporter cells. The system was validated using known chemical inhibitors of the NHEJ pathway, which significantly inhibited the bioluminescent signal generated by CRISPR-Cas9 targeting. The study demonstrated the potential for screening and discovery of NHEJ chemical inhibitors.
There is a need of a non-homologous end joining (NHEJ) pathway reporter system that facilitates screening and discovery of NHEJ chemical inhibitors. In this study, we developed a CRISPR-Cas9 based luciferase turn-on system as a NHEJ pathway reporter. By substituting nucleotide 205C with ATC, we introduced a reading-frame shift and a pre-stop codon into the luciferase coding region and thereby generated a bioluminescent signal mute HEK293T reporter cell line. Then, a CRISPR-Cas9 plasmid expressing a guide RNA targeting luciferase coding region was introduced into the reporter cell line to generate NHEJ-associated indel to restore the reading frame and subsequently turn on the bioluminescent signal. We observed over three-thousand fold increase in signal after CRISPR-Cas9 vector transfection. Different known chemical inhibitors of the NHEJ pathway, such as NU7441, KU0060648, and KU55933, could significantly inhibit the bioluminescent signal generated by CRISPR-Cas9 targeting. In addition, we validated our system by high throughput sequencing.

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