Pieplow Alice, Dastaw Meseret, Sakuma Tetsushi, Sakamoto Naoaki, Yamamoto Takashi, Yajima Mamiko, Oulhen Nathalie, Wessel Gary M
Department of Molecular Biology, Cellular Biology and Biochemistry, Brown University, Providence, RI, 02912, USA.
Ethiopian Biotechnology Institute, Addis Ababa University, NBH1, 4killo King George VI St, Addis Ababa, Ethiopia.
Dev Biol. 2021 Apr;472:85-97. doi: 10.1016/j.ydbio.2021.01.003. Epub 2021 Jan 19.
We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-β signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.
我们试图通过对顺式调控序列进行CRISPR-Cas9编辑来操纵基因功能，而不是对编码区进行更常见的突变。这种方法将使细胞对无义介导的衰变途径或过早终止产生的突变蛋白产物的反应的次级效应最小化。在完全基因敲除会导致致死性的情况下，该策略还能降低基因活性，并且可用于快速鉴定基因表达所必需的关键调控位点。我们在此用已知功能的基因测试了这一策略，作为概念验证，然后将其应用于研究海胆紫球海胆生殖系基因Nanos2的上游基因组区域。我们首先使用CRISPR-Cas9靶向成骨细胞转录因子Alx1和TGF-β信号配体Nodal的既定基因组顺式调控区域，在海胆胚胎中改变这些区域会产生明显的发育缺陷。重要的是，顺式激活位点（Alx1）和顺式抑制位点（Nodal）的突变分别导致预测的转录输出减少和增加。通过基因组突变鉴定出有效的引导RNA后，我们随后使用相同经过验证的引导RNA靶向deadCas9-VP64转录激活因子，以直接增加Nodal转录。最后，我们将这些新方法与更传统的绿色荧光蛋白报告基因构建体方法相结合，以进一步了解Nanos2的转录调控，Nanos2是紫球海胆生殖细胞特性所需的关键基因。通过一系列报告基因检测、上游Cas9-启动子靶向诱变，结合定量PCR和原位RNA杂交，我们得出结论，Nanos2的启动子在海胆胚胎中驱动强烈的mRNA表达，表明其原始生殖细胞（PGC）特异性限制可能反而依赖于转录后调控。总体而言，我们展示了一个由Cas9介导的启动子区域操纵的原理验证工具包，该工具包应适用于大多数使用CRISPR-Cas9的细胞和胚胎。
