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.

我们寻求通过 CRISPR-Cas9 编辑顺式调控序列来操纵基因功能，而不是更典型的编码区突变。这种方法将最大限度地减少细胞反应对无意义介导的衰变途径或过早停止对突变蛋白质产物的二次影响。该策略还允许在完全基因敲除会导致致死的情况下降低基因活性，并且可以应用于快速识别基因表达所必需的关键调控位点。我们在这里用已知功能的基因作为概念证明测试了这一策略，然后将其应用于检查海胆Strongylocentrotus purpuratus中种系基因Nanos2的上游基因组区域. 我们首先使用 CRISPR-Cas9 靶向已建立的成骨细胞转录因子Alx1和 TGF-β 信号配体Nodal的基因组顺式调控区域，这些区域在海胆胚胎中发生改变时会产生明显的发育缺陷。重要的是，顺式激活位点 ( Alx1 ) 和顺式阻遏位点 ( Nodal) 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 gRNA 后，我们随后使用相同的经过验证的 gRNA 靶向 deadCas9-VP64 转录激活因子以直接增加节点转录。最后，我们将这些新方法与更传统的 GFP 报告构建方法配对，以进一步了解 Nanos2 的转录调控，Nanos2是S. purpuratus生殖细胞鉴定所需的关键基因。通过一系列报告基因分析、上游 Cas9 启动子靶向诱变，再加上 qPCR 和原位 RNA 杂交，我们得出结论，Nanos2的启动子驱动海胆胚胎中的强 mRNA 表达，表明其原始生殖细胞 (PGC) 特异性限制可能依赖于转录后调控。总体而言，我们提出了 Cas9 介导的启动子区域操作的原理验证工具包，该工具包应该适用于使用 CRISPR-Cas9 的大多数细胞和胚胎。