Chen Liqun, Zhang Huilian, Zhang Linteng, Li Wenbo, Fan Fengtian, Wu Xiaoyun, Wu Xueling, Lin Jun
College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108 China.
Institute of Apply Genomics, Fuzhou University, Fuzhou, 350108 China.
Cell Mol Bioeng. 2019 Dec 2;13(1):61-72. doi: 10.1007/s12195-019-00606-y. eCollection 2020 Feb.
CRISPR/CAS9 systems, which can be utilized biological experiments, have recently captured much attention for their important roles and benefits. However, full realization of the potential of CRISPR/CAS9 approaches requires addressing many challenges and side effects. The expression of genes and potential side effects of CRISPR/CAS9 in human cells remains to be elucidated. The aim of our study was to explore the effect of CRISPR/CAS9 on gene expression in 293T cells.
A Cas9-expressing PX458 plasmid and Cas9-deactivated PX458-T2A plasmid were used to study the role of CRISPR/CAS9 on regulating gene expression in 293T cells. Gene expression in 293T cells after transfection of the PX458 plasmid or PX458-T2A plasmid was detected by RNA sequencing and correlative statistical analysis. Differential gene expression in both PX458 transfected 293T cells and PX458-T2A transfected 293T cells compared with normal 293T cells was detected using quantitative reverse transcription polymerase chain reaction (RT qPCR). The mRNA and protein levels were measured using reverse transcription PCR and Western blot. Co-IP assay combined with shotgun LC-MS/MS were used to investigate the differences of NGFR-interaction proteins between PX458 transfected 293T cells and PX458-T2A transfected 293T cells.
In this study, we observed that PX458 plasmid transfection and Cas9 expression can affect the expression of different genes, including FOSB (FBJ murine osteosarcoma viral oncogene homolog B), IL-11 (Interleukin-11), MMP1 (matrix metalloproteinase), CYP2D6 (CytochromeP4502D6), and NGFR (matrix metalloproteinase 1). Downregulation of NGFR after PX458 transfection was confirmed by RT qPCR and western blot analysis. NGFR expression was significantly lower in PX458 transfected 293T cells than in normal 293T cells and PX458-T2A transfected 293T cells. The co-IP dilutions analyzed by shotgun LC-MS/MS showed a total of 183 proteins interact with NGFR in PX458 transfected 293T cells while 221 proteins interact with NGFR were identified in PX458-T2A transfected 293T cells using the MASCOT engine.
Cas9 expression by transfection of the PX458 plasmid was negatively correlated with the NGFR mRNA level and NGFR protein expression in 293T cells, while PX458-T2A, in which Cas9 is deactivated, did not affect NGFR expression. The decrease in NGFR expression also affects the amount of proteins that interact with NGFR. These results suggest that the effect of Cas9 on NGFR expression and the expression of other genes should be noticed when developing cell-based studies and therapies utilizing CRISPR/CAS9 systems.
CRISPR/CAS9系统可用于生物实验，因其重要作用和优势，近来备受关注。然而，要充分发挥CRISPR/CAS9方法的潜力，需要应对诸多挑战和副作用。CRISPR/CAS9在人类细胞中的基因表达及潜在副作用仍有待阐明。我们研究的目的是探索CRISPR/CAS9对293T细胞中基因表达的影响。
使用表达Cas9的PX458质粒和失活Cas9的PX458-T2A质粒，研究CRISPR/CAS9在调控293T细胞基因表达中的作用。通过RNA测序和相关统计分析，检测转染PX458质粒或PX458-T2A质粒后293T细胞中的基因表达。使用定量逆转录聚合酶链反应（RT qPCR），检测与正常293T细胞相比，PX458转染的293T细胞和PX458-T2A转染的293T细胞中的差异基因表达。使用逆转录PCR和蛋白质印迹法测量mRNA和蛋白质水平。采用免疫共沉淀（Co-IP）试验结合鸟枪法液相色谱-串联质谱（LC-MS/MS），研究PX458转染的293T细胞和PX458-T2A转染的293T细胞中NGFR相互作用蛋白的差异。
在本研究中，我们观察到PX458质粒转染和Cas9表达可影响不同基因的表达，包括FOSB（FBJ小鼠骨肉瘤病毒癌基因同源物B）、IL-11（白细胞介素-11）、MMP1（基质金属蛋白酶）、CYP2D6（细胞色素P450 2D6）和NGFR（神经生长因子）。RT qPCR和蛋白质印迹分析证实了PX458转染后NGFR的下调。PX458转染的293T细胞中NGFR表达显著低于正常293T细胞和PX458-T2A转染的293T细胞。使用MASCOT引擎，通过鸟枪法LC-MS/MS分析免疫共沉淀稀释液，结果显示在PX458转染的293T细胞中有183种蛋白质与NGFR相互作用，而在PX458-T2A转染的293T细胞中有221种蛋白质与NGFR相互作用。
转染PX458质粒导致的Cas9表达与293T细胞中NGFR mRNA水平和NGFR蛋白表达呈负相关，而失活Cas9的PX458-T2A则不影响NGFR表达。NGFR表达的降低也会影响与NGFR相互作用的蛋白质数量。这些结果表明，在开展利用CRISPR/CAS9系统的细胞研究和治疗时，应注意Cas9对NGFR表达及其他基因表达的影响。
