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Robustness of Catalytically Dead Cas9 Activators in Human Pluripotent and Mesenchymal Stem Cells.
Molecular Therapy - Nucleic Acids ( IF 8.8 ) Pub Date : 2020-02-27 , DOI: 10.1016/j.omtn.2020.02.009
Paolo Petazzi 1 , Raul Torres-Ruiz 2 , Antonella Fidanza 3 , Heleia Roca-Ho 1 , Francisco Gutierrez-Agüera 1 , Julio Castaño 1 , Sandra Rodriguez-Perales 4 , Rafael Díaz de la Guardia 1 , Belén López-Millán 1 , Anna Bigas 5 , Lesley M Forrester 3 , Clara Bueno 6 , Pablo Menéndez 7
Affiliation  

Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.



中文翻译:

人多能和间充质干细胞中催化死亡的Cas9活化剂的稳健性。

人多能干细胞(hPSC)和间充质基质/干细胞(hMSC)是细胞疗法以及人类发育和疾病建模的临床相关来源。许多基于干细胞的应用都依赖于同时激活多个内源基因来修饰细胞命运的能力。然而,这些细胞的遗传干预仍然具有挑战性。与特定激活域融合的几种催化死亡的Cas9(dCas9)蛋白在通过特定的单向导RNA(sgRNA)定向到其调节区域时,可以调节基因表达。在这项研究中,我们比较了第一代dCas9-VP64激活剂和第二代系统dCas9-SAM和dCas9-SunTag在hPSC和hMSC中诱导基因表达的能力。测试了几种干细胞系的单基因和多重基因激活。当比较几个基因的激活时,所有三个系统都在单个和多重设置下诱导了特异性和有效的基因表达,但是dCas9-SAM和dCas9-SunTag系统导致了最高和最一致的基因表达水平。用多个sgRNA同时靶向同一基因不会导致hPSC和hMSC中基因表达的累加水平。我们证明了第二代dCas9激活剂的健壮性和特异性,可同时激活临床相关的人类干细胞中的多个内源基因。用多个sgRNA同时靶向同一基因不会导致hPSC和hMSC中基因表达的累加水平。我们证明了第二代dCas9激活剂的健壮性和特异性,可同时激活临床相关的人类干细胞中的多个内源基因。用多个sgRNA同时靶向同一基因不会导致hPSC和hMSC中基因表达的累加水平。我们证明了第二代dCas9激活剂的健壮性和特异性,可同时激活临床相关的人类干细胞中的多个内源基因。

更新日期:2020-02-27
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