Zhang Shuyan, Luo Tao, Wang Jinke
State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, People's Republic of China.
J Inflamm Res. 2021 Mar 17;14:917-928. doi: 10.2147/JIR.S298938. eCollection 2021.
NF-κB is a sequence-specific DNA-binding transcription factor that plays key roles in inflammation and cancer. It is well known that NF-κB is over-activated in these diseases. NF-κB inhibitors are therefore developed as promising drugs for these diseases. However, finding NF-κB inhibitors is dependent on effective screening platforms.
For providing an easy and visualizable tool for screening NF-κB inhibitors, and other NF-κB-related studies, this study edited all five genes of NF-κB family (RELA, RELB, CREL, NF-κB1, NF-κB2) in three different cell lines (293T, HepG2, and PANC1) with both TALEN and CRISPR. The edited NF-κB genes were repaired by homology-dependent repair using a linear homologous donor containing ZsGreen coding sequence. The edit efficiency was thus directly evaluated by detecting cellular fluorescence. The editing efficiency was also confirmed by PCR detection of NF-κB-ZsGreen fused genes.
It was found that all genes were more efficiently edited by TALEN in all cells than CRISPR. The positive cells were then isolated from the TALEN-edited cell pool by flow cytometry. The purified positive cells were finally evaluated by regulating NF-κB activity with a known NF-κB inhibitor, BAY 11-7082, and an NF-κB-targeting artificial microRNA, miR533. The results revealed that all the labeled NF-κB genes responded well to the two kinds of NF-κB activity regulators in all cell lines.
This study thus obtained 15 cell lines with NF-κB-ZsGreen fused genes, which provide an easy and visualizable tool for screening NF-κB inhibitors and other NF-κB-related studies.
核因子κB（NF-κB）是一种序列特异性DNA结合转录因子，在炎症和癌症中起关键作用。众所周知，NF-κB在这些疾病中过度激活。因此，NF-κB抑制剂被开发为治疗这些疾病的有前景的药物。然而，寻找NF-κB抑制剂依赖于有效的筛选平台。
为了提供一种用于筛选NF-κB抑制剂以及其他与NF-κB相关研究的简便且可视化的工具，本研究利用转录激活样效应因子核酸酶（TALEN）和规律成簇间隔短回文重复序列（CRISPR）在三种不同细胞系（293T、HepG2和PANC1）中编辑NF-κB家族的所有五个基因（RELA、RELB、CREL、NF-κB1、NF-κB2）。使用含有ZsGreen编码序列的线性同源供体通过同源依赖性修复来修复编辑后的NF-κB基因。因此，通过检测细胞荧光直接评估编辑效率。还通过PCR检测NF-κB-ZsGreen融合基因来确认编辑效率。
发现在所有细胞中，TALEN对所有基因的编辑效率均高于CRISPR。然后通过流式细胞术从TALEN编辑的细胞池中分离出阳性细胞。最后，用已知的NF-κB抑制剂BAY 11-7082和靶向NF-κB的人工微小RNA miR533调节NF-κB活性，对纯化的阳性细胞进行评估。结果显示，在所有细胞系中，所有标记的NF-κB基因对这两种NF-κB活性调节剂均反应良好。
本研究由此获得了15个带有NF-κB-ZsGreen融合基因的细胞系，为筛选NF-κB抑制剂以及其他与NF-κB相关研究提供了一种简便且可视化的工具。
