Han Jeong Hoon, Lee Hong Jo, Kim Tae Hyun
Department of Animal Science, The Pennsylvania State University, University Park, PA, United States.
Division of Animal Sciences, University of Missouri, Columbia, MO, United States.
Front Genome Ed. 2023 Oct 25;5:1269115. doi: 10.3389/fgeed.2023.1269115. eCollection 2023.
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 (, and ) 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.
DNA调控元件精确控制基因何时、何地以及如何被激活。因此，了解这些元件的功能可以揭示基因调控网络的复杂性。全基因组范围内的显著变异主要存在于DNA的非编码区域，所以理解预测的功能调控元件对于理解这些基因组标记的生物学背景至关重要，这些标记可纳入育种计划。CRISPR技术的出现为研究基因组中的非编码调控元件提供了强大工具。在本研究中，我们利用动物基因组功能注释项目的表观遗传数据，在鸡基因组中鉴定与三个基因（此处原文缺失基因名称）相关的启动子和假定的增强子区域。为了鉴定增强子区域，我们设计了靶向启动子和候选增强子区域的引导RNA，并在鸡DF-1细胞中利用dCas9-p300和dCas9-VPR作为转录激活剂的CRISPR激活（CRISPRa）。通过比较启动子激活以及启动子与假定增强子共同激活时目标基因的表达水平，我们能够鉴定出表现出增强上调的功能性增强子。总之，我们的数据表明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，突出了其在非编码区域功能验证方面的潜力。
