School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou, 510006, China.
Arch Toxicol. 2018 Oct;92(10):3103-3115. doi: 10.1007/s00204-018-2287-8. Epub 2018 Aug 21.
Reporter cell lines are a particularly useful tool to screen for the skin sensitization potential of chemicals. Current cell models based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids. However, plasmid-based reporters may ignore comprehensive aspects of induction, thus affecting the accuracy of hazard identification. Herein, we developed a novel HaCaT-based reporter system, EndoSens, whereby luciferase was specifically inserted into the cassette for heme oxygenase (decycling) 1 (HMOX1, the most consistent marker induced by skin sensitizers) by CRISPR/Cas9. Testing data from 20 coded substances showed an accuracy of 90%, sensitivity of 91.7%, and specificity of 87.5%, which exceeded the OECD requirement. Among the 35 chemicals examined, predictivity was better than reported for the validated KeratinoSens™. These results indicate that the EndoSens assay could advance the predictivity of skin sensitization, thus making it a promising tool for in vitro skin sensitization testing.
报道细胞系是筛选化学物质皮肤致敏潜能的一种特别有用的工具。目前基于 Keap1-Nrf2 的细胞模型通过抗氧化反应元件-荧光素酶质粒来模拟诱导。然而，基于质粒的报告器可能会忽略诱导的综合方面，从而影响危险识别的准确性。在此，我们通过 CRISPR/Cas9 开发了一种新型的 HaCaT 报道系统 EndoSens，其中荧光素酶通过 CRISPR/Cas9 被特异性插入到血红素加氧酶（脱环）1（HMOX1，最一致的皮肤致敏剂诱导标志物）的盒中。来自 20 种编码物质的测试数据显示，准确性为 90%，灵敏度为 91.7%，特异性为 87.5%，超过了 OECD 的要求。在检查的 35 种化学物质中，预测性优于经验证的 KeratinoSens™的报道。这些结果表明，EndoSens 测定法可以提高皮肤致敏的预测性，从而使其成为体外皮肤致敏测试的有前途的工具。
