Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.
Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.
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作者：
内源性基因特异性调控技术对基因功能的研究具有重要价值，并在治疗学方面具有巨大潜力。我们创建了CRISPR-on系统，这是一种双组分转录激活因子，由与转录激活结构域融合的核酸酶死亡Cas9（dCas 9）蛋白和具有与基因启动子互补序列的单向导RNA（sgRNA）组成。我们证明了CRISPR-on可以以可调的方式有效地激活人类和小鼠细胞中的外源报告基因。此外，我们表明，强大的报告基因在体内激活可以通过注射到小鼠受精卵的系统组件。此外，我们表明CRISPR-on可以激活内源性IL 1 RN，SOX 2和OCT 4基因。最有效的基因激活是通过与近端启动子结合的3-4个sgRNA簇实现的，表明它们在基因诱导中的协同作用。值得注意的是，当靶向多个基因的sgRNA同时引入细胞中时，实现了稳健的多重内源基因激活。全基因组表达谱分析表明该系统具有高度的特异性。
Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.
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影响因子：
46.9
作者：
通讯作者：
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影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
DOI：
10.1073/pnas.040552697
发表时间：
2000-02-15
影响因子：
11.1
作者：
Beerli, RR;Dreier, B;Barbas, CF
通讯作者：
Barbas, CF
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
46.9
作者：
通讯作者：
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