Generation of Endogenous Promoter-Driven Luciferase Reporter System Using CRISPR/Cas9 for Investigating Transcriptional Regulation of the Core Clock Gene BMAL1

Abstract

Humans and other organisms are continuously exposed to thousands of chemicals through the atmosphere, drinking water, food, or direct contact. A large proportion of such chemicals are present in very low concentrations and may have synergistic effects, even at their no-observed-adverse-effect level (NOAEL). Complex mixtures of contaminants are very difficult to assess by traditional toxicological methods. There is increasing attention on how different pollutants induce adverse physiological functions in the human body through effects on the circadian rhythm. However, it is very difficult to screen for compounds with circadian-rhythm-disrupting effects from a large number of chemicals or their complex mixtures. We established a stable firefly luciferase reporter gene knock-in U2-OS cell line by CRISPR/Cas9 to screen circadian-rhythm-disrupting pollutants. The luciferase gene was inserted downstream of the core clock gene BMAL1 and controlled by an endogenous promoter. Compared to detection systems using exogenous promoters, these cells enable the detection of compounds that interfere with the circadian rhythm system mediated by BMAL1 gene expression. The U2-OS knock-in cells showed BMAL1 and luciferase activity had parallel changes when treated with BMAL1 inhibitor and activator. Furthermore, the luciferase reporter gene has high sensitivity and is faster and more cost-effective than classic toxicology methods. The knock-in cell line can be used for high-throughput and efficient screening of circadian-rhythm-disrupting chemicals such as drugs and pollutants.

Target-triggered CRISPR–Cas13a autocatalysis-driven amplification strategy for one-step detection of circadian clock gene

“…8 Moreover, BMAL1 has been found to modulate the response of cancer cells to chemotherapy and radiation therapy, highlighting the importance of BMAL1 regulation in cancer diagnosis and treatment. 9,10 Therefore, the accurate detection of BMAL1 in cancer cells may provide valuable insights into the molecular mechanisms underlying cancer development and progression, as well as potential targets for cancer therapy. Currently, BMAL1 mRNA was mainly identified based on polymerase chain reaction (PCR), 11,12 northern blotting, 13 microarray, 14 and RNA sequencing.…”