不稳定EGFP细胞模型的构建及其在基因编辑体系评价中的应用*

目的:为了更好地评价基因编辑效率,满足高通量筛选应用中快速、高效的检测要求,在细胞上建立一个原位检测方法具有重要的意义。通过检测荧光蛋白信号强度的变化可以评价CRISPR系统在细胞中的基因编辑情况,然而这一方法的效率受限于荧光蛋白较长的半衰期。方法:将鸟氨酸脱羧酶降解结构域(含PEST序列)与EGFP融合,通过慢病毒系统感染HEK-293T细胞,获得了表达单拷贝、EGFP-PEST报告基因的稳转细胞系。结果:与EGFP相比,EGFP-PEST在细胞内的降解速度明显加快,荧光水平在4 h内显著降低。利用该模型比较了3种商品化脂质体介导的CRISPR/Cas9基因编辑效率,能够在2~4 d实现定性和定量评价。结论:这一模型能够快速、灵敏地指示基因编辑效果,可以用于不同CRISPR系统或新递送工具的高通量筛选和评价。

Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation

Objective: To meet the requirements of rapid and efficient gene editing detection in high-throughput screening applications, it is of great significance to establish an in situ evaluation method on cells. EGFP can be used to evaluate the gene editing performance mediated by the CRISPR system, but the efficiency is limited by the long half-life of EGFP. Methods: A version of destabilized EGFP (EGFP-PEST) was constructed by fusing degradation domain of ornithine decarboxylase (containing PEST motif) to EGFP. The EGFP-PEST gene was introduced into the chromosome of HEK-293T cells by lentivirus, and the copy number of EGFP-PEST gene was measured by RT-qPCR. Finally, cell strains with single copy of EGFP-PEST transgene were established. Results: Compared to unmodified EGFP, the cellular fluorescence of EGFP-PEST significantly decreased within 4 h, suggesting efficient PEST-mediated protein degradation. The cell model was used to evaluate the potential of three commercial lipids to deliver CRISPR/Cas9 complex. Data showed that gene editing was detected in 2-4 days by quantitative or qualitative measurements. Conclusion: This cell model can be used in high-throughput screening for new CRISPR tools or novel delivery systems by indicating the gene editing rapidly and sensitively.