Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks. Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation. All lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not. Baseline expression of both cytokines was not different between the lines regardless of CFTR genotype. All lines responded to TNFa and IL1b by increasing IL6 and CXCL8 (IL8) mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNFa, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR’s absence, others required stimulation for differences to be apparent. Together, these data suggest cells respond to CFTR’s absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR’s absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation. Overall design: Compare cells with intact CFTR to cells lacking CFTR for overall gene expression under basal and TNFa-stimulated conditions

囊性纤维化（cystic fibrosis, CF）患者可出现多器官炎症水平升高，但其究竟是CF细胞的固有特征，还是促炎环境所导致的结果，目前尚不明确。本研究通过CRISPR/Cas9介导的囊性纤维化跨膜传导调节因子（cystic fibrosis transmembrane conductance regulator, CFTR）基因突变，从Caco-2细胞中构建得到17株亚克隆细胞系。将携带功能性CFTR的克隆细胞系（CFTR+）与不携带功能性CFTR的克隆细胞系（CFTR-）进行对比，以直接明确CFTR在炎症基因调控中的作用。所有细胞系均保留CFTR mRNA的表达以及紧密连接的形成能力。CFTR+细胞系可在佛司可林（forskolin）刺激下产生短路电流，而CFTR-细胞系则无此现象。无论CFTR基因型如何，各细胞系的两种细胞因子基线表达水平均无显著差异。所有细胞系在肿瘤坏死因子α（tumor necrosis factor alpha, TNFα）和白细胞介素1β（interleukin 1 beta, IL1β）刺激下，均可上调白细胞介素6（interleukin 6, IL6）和趋化因子配体8（C-X-C motif chemokine ligand 8, CXCL8，又称IL8）的mRNA水平；但CFTR-细胞系的CXCL8 mRNA表达量显著高于CFTR+细胞系。本研究对6株CFTR-细胞系和6株CFTR+细胞系在TNFα刺激前后的转录组进行比较，以分析CFTR基因型对基因差异表达的影响。结果显示，部分基因的差异表达仅由CFTR的缺失所导致，而另有部分基因的差异表达则需在刺激条件下才会显现。综合以上数据，提示细胞可通过调控转录网络以响应CFTR的缺失，其中部分转录网络仅在细胞暴露于炎症等不同环境背景时才会显现功能差异。针对炎症反应而言，本研究数据支持如下模型：CFTR的缺失会使细胞进入预激活的促炎状态，该状态仅在受到外界刺激时才会显现。实验整体设计：在基线条件与TNFα刺激条件下，分别比较CFTR完整的细胞与CFTR缺失的细胞的全基因表达谱。