Abrahimi Parwiz, Chang William G, Kluger Martin S, Qyang Yibing, Tellides George, Saltzman W Mark, Pober Jordan S
From the Department of Immunobiology (P.A., M.S.K., J.S.P.), Department of Internal Medicine (W.G.C., Y.Q.), Department of Surgery (G.T.), and Department of Biomedical Engineering (W.M.S.), Yale University, New Haven, CT.
Circ Res. 2015 Jul 3;117(2):121-8. doi: 10.1161/CIRCRESAHA.117.306290. Epub 2015 May 4.
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)/CRISPR-associated protein 9 nuclease (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 biallelic gene disruption in untransformed human EC.
To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator 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 reintroduction of class II transactivator expression.
CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.
内皮细胞（EC）参与许多生理和病理过程，目前广泛使用人EC培养物对其进行建模，但对这些未转化细胞进行基因操作在技术上具有挑战性。成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9核酸酶（Cas9）技术提供了一种有前景的新方法。然而，诱变的培养细胞需要克隆才能产生同质群体，而高度分化的人EC有限的复制寿命对此形成了障碍。
创建一种使用CRISPR/Cas9在未转化的人EC中产生双等位基因破坏的简单但高效的方法。
为了证明原理，我们使用CRISPR/Cas9破坏II类反式激活因子基因。我们使用内皮集落形成细胞衍生的EC和慢病毒载体来递送CRISPR/Cas9元件，以消除II类主要组织相容性复合体分子的EC表达，进而消除激活同种异体CD4(+) T细胞的能力。我们表明观察到的功能丧失源于II类反式激活因子的双等位基因破坏，而细胞的其他基本特性保持完整，包括在体内自组装成血管，并且通过重新引入II类反式激活因子表达可以挽救改变的表型。
CRISPR/Cas9修饰的人EC为血管研究和再生医学/组织工程提供了一个强大的平台。
