Epigenome editing of the CFTR-locus for treatment of cystic fibrosis.
Epigenome editing of the CFTR-locus for treatment of cystic fibrosis.
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DOI：
10.1016/j.jcf.2021.04.008
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发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Reddy TE
中科院分区：
文献类型：
--
作者：
Kabadi AM;Machlin L;Dalal N;Lee RE;McDowell I;Shah NN;Drowley L;Randell SH;Reddy TE
控制CFTR基因在整个身体中表达多样性的机制是复杂的。多个内含子和远端调节元件负责调节跨组织的差异CFTR表达。利用已发表的数据，使用基于CRISPR/dCas 9的表观基因组编辑工具鉴定并询问了18个高优先级基因组区域的CFTR增强子功能。通过dCas 9 p300和dCas 9 KRAB分别评估每个区域增强或抑制CFTR表达的能力。使用CRISPR/dCas 9表观基因组编辑测试多个基因组区域的增强子活性。当靶向CFTR低表达细胞系中的启动子和转录起始位点上游44 kb的区域时，dCas 9 p300介导CFTR mRNA水平的显著增加。靶向启动子的多种gRNA诱导CFTR蛋白水平的稳健增加。相比之下，dCas 9 KRAB介导的阻遏更加稳健，18个评估的基因组区域中有10个诱导CFTR蛋白敲低。为了评估调节CFTR基因调控的治疗功效，使用dCas 9 p300从ΔF508/ΔF508人支气管上皮细胞中的内源基因座诱导升高水平的CFTR。Ussing室研究表明，响应于CRISPR诱导的ΔF508 CFTR mRNA表达沿着VX 809处理，离子转运协同增加。基于CRISPR/dCas 9的表观基因组编辑为询问CFTR增强子功能提供了以前未探索的工具。在这里，我们证明了增加CFTR表达的治疗干预可以提高CFTR调节剂的功效。更好地理解CFTR调节机制可以为囊性纤维化疗法的开发发现新的治疗干预措施。
Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues. Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9p300 and dCas9KRAB for its ability to enhance or repress CFTR expression, respectively. Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9p300 mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9KRAB-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9p300 was used to induce elevated levels of CFTR from the endogenous locus in ΔF508/ΔF508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of ΔF508 CFTR mRNA along with VX809 treatment. CRISPR/dCas9-based epigenome editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.
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