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Published on: June 1, 2014
Development of a CRISPR-Cas9 Based Luciferase Turn-On System as Nonhomologous End Joining Pathway Reporter
Yi Wang1, Yanjie Zhao1, Weijun Su2
1Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin, 300060, P. R. China.
Abstract:
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.
Insights
Researchers developed a CRISPR-Cas9 based reporter system to screen for non-homologous end joining (NHEJ) inhibitors. This novel system enables efficient discovery of chemical compounds targeting the NHEJ DNA repair pathway.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The non-homologous end joining (NHEJ) pathway is crucial for DNA double-strand break repair.
- There is a significant need for robust reporter systems to facilitate the screening and discovery of NHEJ chemical inhibitors.
- Existing methods may lack the sensitivity or throughput required for comprehensive inhibitor screening.
Purpose of the Study:
- To develop and validate a novel CRISPR-Cas9 based luciferase reporter system for monitoring NHEJ pathway activity.
- To enable efficient screening and discovery of small molecule inhibitors targeting the NHEJ pathway.
- To provide a sensitive and high-throughput platform for studying DNA repair mechanisms.
Main Methods:
- A HEK293T reporter cell line was engineered with a luciferase gene containing a frameshift mutation and a pre-stop codon, rendering it bioluminescent signal-mute.
- CRISPR-Cas9 technology was employed to introduce targeted double-strand breaks, inducing NHEJ-mediated repair and restoring the luciferase reading frame, thereby activating bioluminescence.
- The system's functionality was validated using known NHEJ inhibitors (e.g., NU7441, KU0060648, KU55933) and confirmed through high-throughput sequencing.
Main Results:
- The CRISPR-Cas9 system successfully activated bioluminescence in the reporter cell line, with over a three-thousand-fold signal increase observed upon NHEJ pathway activation.
- Treatment with known NHEJ inhibitors significantly suppressed the CRISPR-Cas9-induced bioluminescent signal, confirming the reporter's responsiveness to pathway modulation.
- High-throughput sequencing validated the NHEJ-mediated indel formation responsible for restoring the luciferase reading frame.
Conclusions:
- A novel and effective CRISPR-Cas9 based turn-on luciferase reporter system for the non-homologous end joining pathway has been successfully developed.
- This system provides a sensitive, quantifiable, and high-throughput method for screening chemical inhibitors of NHEJ.
- The validated reporter system holds significant potential for advancing the discovery of therapeutics targeting DNA repair pathways.
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