Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.
Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.
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DOI：
10.1161/circresaha.117.306290
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发表时间：
2015-07-03
影响因子：
20.1
通讯作者：
Pober JS
中科院分区：
文献类型：
--
作者：
Abrahimi P;Chang WG;Kluger MS;Qyang Y;Tellides G;Saltzman WM;Pober JS
内皮细胞（EC）参与许多生理和病理过程被广泛使用人类EC培养物建模，但这些未转化细胞的遗传操作在技术上具有挑战性。规则间隔短回文重复序列（CRISPR）/Cas9技术提供了一种有前途的新方法。然而，诱变培养的细胞需要克隆以产生同质群体，并且分化良好的人EC的有限复制寿命为这样做提供了障碍。创建一种简单但高效的方法，使用CRISPR/Cas9在未转化的人EC中产生双等位基因基因破坏。为了证明原理，我们使用CRISPR/Cas9来破坏II类反式激活因子（CIITA）的基因。我们使用内皮集落形成细胞（ECFC）衍生的EC和慢病毒载体来递送CRISPR/Cas9元件，以消除II类MHC分子的EC表达，并与之一起激活同种异体CD 4 + T细胞的能力。我们发现，观察到的功能丧失来自CIITA中的双等位基因基因破坏，使细胞的其他基本特性保持完整，包括体内自组装成血管，并且改变的表型可以通过重新引入CIITA表达来挽救。CRISPR/Cas9修饰的人类EC为血管研究和再生医学/组织工程提供了强大的平台。
The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. To create a simple but highly efficient method using CRISPR/Cas9 to generate bi-allelic gene disruption in untransformed human EC. To demonstrate proof-of-principle we used CRISPR/Cas9 to disrupt the gene for the class II transactivator (CIITA). We used endothelial colony forming cell (ECFC)-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II MHC molecules and with it, the capacity to activate allogeneic CD4+ T cells. We show the observed loss-of-function arises from bi-allelic gene disruption in CIITA that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by re-introduction of CIITA expression. CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.