NEW METHODS IN CARDIOVASCULAR BIOLOGY Efficient Gene Disruption in Cultured Primary Human Endothelial Cells by CRISPR/Cas9

Subject codes: [161] Transplantation [142] Gene Expression [97] Other Vascular biology [95] Endothelium/vascular type/nitric oxide ABSTRACT Rationale: 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. Objective: To create a simple but highly efficient method using CRISPR/Cas9 to generate bi-allelic gene disruption in untransformed human EC. Methods and Results: 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. Conclusions: CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering. Nonstandard Abbreviations and Acronyms: Cas9 CRISPR-associated protein 9 nuclease CRISPR clustered regularly interspaced short palindromic repeats CIITA class II transactivator EC endothelial cell ECFC endothelial colony forming cell HLA human leukocyte antigen HUVEC human umbilical vein endothelial cell (IFN)-γ interferon gamma IL-2 interleukin 2 LFA-3 lymphocyte function-associated antigen-3 MHC major histocompatibility complex sgRNA single guide RNA TEER transendothelial electrical resistance