基于CRISPR/Cas9基因定点敲入技术利用荧光标记斑马鱼cyp1a基因及其在PAHs检测上的应用

通过测定鱼类cyp1a基因表达量来监测石油类、PAHs等有机污染是目前最为有效的生物标志物监测技术。我们前期构建了Tg(cyp1a:mCherry)转基因荧光斑马鱼，并利用环境样品进行暴露分析，结果发现暴露后斑马鱼荧光强度和样品中PAHs含量具有良好的相关性；但亦发现这种利用随机插入的方法构建的荧光斑马鱼其插入位点和拷贝数直接影响荧光的表达效率，不能真实反映cyp1a基因的表达情况。因此本研究拟利用CRISPR/Cas9基因编辑技术，将不同拷贝数的外源荧光基因定点敲入到斑马鱼内源cyp1a基因启动子之后，构建基因敲入的荧光斑马鱼，并分析荧光基因与其自身cyp1a基因的表达差异；同时利用环境样品对其进行暴露验证，分析样品中PAHs含量和荧光强度之间的相关性。旨在建立一种直观、经济的PAHs生物监测方法，以此提高环境监管效率，节约人力和成本，这对保护环境和保障人民健康具有重要的意义！

The quantification of cyp1a expression in fish is the most effective method so far to identify PAHs contamination in the aquatic ecosystem. Previously, we not only generated a transgenic zebrafish line Tg(cyp1a:mCherry) with cyp1a promoter driving mCherry expression but also precisely monitor PAHs contamination in different environmental samples using newly established zebrafish. Further, our results showed that the response of mCherry was consistent with the PAHs exposure concentrations. However, the insertion site and copy number of mCherry directly affected its expression efficiency, therefore, mCherry responses not truly reflected the expression of cyp1a gene. Here, we will use CRISPR/Cas9 technique to generate a transgenic zebrafish line by inserting the fluorescent tag after the cyp1a promoter and analyze whether the expression of mCherry is consistent with the endogenous cyp1a gene expression. We will also evaluate the sensitivity of the newly generated zebrafish line by exposing it to the environmental samples in order to test whether it can be used as an In vivo assay for accurate monitoring of PAHs in the environment. In a nutshell, we plan to modify our previously established zebrafish line for precise measurement of PAHs contamination via more sensitive, efficient, and convenient method, which will be of great significance to safeguard environment and human health.