PDF(Pubmed)
Abstract：
DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (HBBA, IRF7, and PPARG) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.
摘要：
DNA调控元件错综复杂地控制着，where,以及基因是如何被激活的。因此,了解这些元素的功能可以揭示遗传调控网络的复杂性。全基因组显著变异主要存在于DNA的非编码区，因此，理解预测的功能调节元件对于理解这些基因组标记的生物学背景至关重要，可以纳入育种计划。CRISPR技术的出现为研究基因组中的非编码调控元件提供了有力的工具。在这项研究中,我们利用来自动物基因组功能注释项目的表观遗传数据来鉴定与三个基因相关的启动子和推定增强子区域(HBBA，鸡基因组中的IRF7和PPARG）。为了识别增强子区域，我们设计了靶向启动子和候选增强子区域的指导RNA,并利用CRISPR激活(CRISPRa)与dCas9-p300和dCas9-VPR作为鸡DF-1细胞中的转录激活因子.通过比较启动子激活和启动子与推定增强子共激活的靶基因表达水平,我们能够鉴定出表现出增强的上调的功能增强剂。总之,我们的发现证明了CRISPRa通过靶向鸡基因组中的调控元件精确操纵内源基因表达的显著效率,突出了其对非编码区功能验证的潜力。
