CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin.
CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin.
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DOI：
10.1016/j.ydbio.2021.01.003
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发表时间：
2021-04
影响因子：
2.7
通讯作者：
Wessel GM
中科院分区：
文献类型：
--
作者：
Pieplow A;Dastaw M;Sakuma T;Sakamoto N;Yamamoto T;Yajima M;Oulhen N;Wessel GM
我们试图通过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对无义介导的衰变途径或突变蛋白产物的过早停止反应的继发性影响。这种策略还允许在完全基因敲除会导致致命的情况下降低基因活性，并且它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来测试这一策略，作为概念的证明，然后将其应用于海胆种系基因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）和式抑制位点（Nodal）的突变分别导致预期的转录输出减少和增加。在通过基因组突变鉴定出有效的grna后，我们使用相同的经过验证的grna靶向死cas9 - vp64转录激活子来直接增加Nodal转录。最后，我们将这些新方法与更传统的GFP报告基因构建方法结合起来，进一步了解紫癜性葡萄球菌生殖细胞鉴定所需的关键基因Nanos2的转录调控。通过一系列报告基因实验、上游cas9启动子靶向诱变、qPCR和原位RNA杂交，我们得出结论，Nanos2启动子在海胆胚胎中驱动mRNA的强表达，表明其对原始生殖细胞（PGC）特异性的限制可能依赖于转录后调控。总的来说，我们提出了一个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。
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 test 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 it’s 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.
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影响因子：
14.9
作者：
Cameron RA;Samanta M;Yuan A;He D;Davidson E
通讯作者：
Davidson E
影响因子：
64.8
作者：
Kobayashi, S;Yamada, M;Kitamura, T
通讯作者：
Kitamura, T
DOI：
10.1073/pnas.95.22.13062
发表时间：
1998-10-27
影响因子：
11.1
作者：
Arenas-Mena, C;Martinez, P;Davidson, EH
通讯作者：
Davidson, EH
影响因子：
2.7
作者：
Duboc, Veronique;Lapraz, Francois;Lepage, Thierry
通讯作者：
Lepage, Thierry
影响因子：
4.6
作者：
Fresques, Tara M.;Wessel, Gary M.
通讯作者：
Wessel, Gary M.