Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9

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)/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.

Objective: To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic 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. 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.

Conclusions: CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.

Keywords: clustered regularly interspaced short palindromic repeats; endothelial cells; genetic engineering; genetic techniques; immunologic techniques.

Figure 1. High efficiency disruption of CIITA by CRISPR/Cas9 in human EC

(A) Unmodified EC stimulated with IFN-γ increase expression of MHC class I (HLA-A,B,C) and class II (HLA-DR), whereas a high proportion of EC transduced with TetOn-Cas9/sgCIITA vectors upregulate MHC class I but show either reduced (HLA-DR^mid) or complete loss of MHC class II (HLA-DR^neg) expression, (B) qRT-PCR analysis of FACS isolated WT and HLA-DR^neg EC for CIITA, HLA-DRA, and CXCL10 transcripts, (C) HLA-DR^neg EC clonally sorted by single-cell FACS, expanded, and sequenced across sgRNA target site demonstrates bi-allelic indel mutations in CIITA but not in the likeliest off-target site (SLC6A9). Representative plots shown from multiple donors from 3 independent experiments.

Figure 2. CIITA^null EC retain their characteristic endothelial identity

(A) FACS isolated CIITA^null EC expanded for two weeks remain refractory to IFN-γ-induced upregulation of MHC II but are otherwise indistinguishable from unmodified (WT) EC with respect to surface marker expression (PECAM-1, blood group H antigen), (B) viability, (C) formation of VE-cadherin positive cell-cell lateral borders, and (D) characteristic TNF-α and IFN-γ responses pooled from 3 independent donors. Scale bars 50 µm.

Figure 3. CIITA^null EC retain ability to form vessels in vitro and in vivo

(A) CIITA^null EC, like unmodified (WT) EC, spontaneously assemble into cord-like structures (outlined) in 3-D culture in vitro at 24h, (B) CIITA^null EC form perfused vessels (arrows) as detected by H&E that are lined by human EC (identified by Ab reactive with human CD31 and HLA-A,B,C with insets of murine vessels to show species specificity) and that recruit host (smooth muscle α-actin expressing) mural cells when implanted in SCID/bg mice like unmodified WT EC and (C) like EC in vitro, perfused vessels formed from CIITAnull EC are refractory to IFN-γ induced expression of MHC II in vivo as detected by immunofluorescence. Representative figures and mean ± SD from (a) 4 gels, (b and c) 9 mice bearing WT and CIITAnull implants. Scale bar (a) 25 µM and (b and c) 50 µM.

Figure 4. Loss of the ability of CIITA^null EC to activate allogeneic CD4+ memory T cells is rescued by CIITA transduction

(A) Expression of class II MHC molecules in CIITA^null EC is rescued upon reintroduction of functional copy of CIITA by retroviral transduction. CIITA^null EC lose ability to activate alloreactive memory CD4+ T cells, which is rescued with CIITA retrovirus, as measured by (B) quantitative ELISA for IL-2 and IFN-γ produced in 24h supernatants, as well as (C) CFSE dilution and HLA-DR activation markers at 7d. Representative plots and mean ± SD from n=4 replicates. Similar results were seen in 3 independent experiments. LZRS is empty retroviral expression vector. * in (B) and (C) indicates statistically significant difference by post-hoc Bonferroni correction.