HIF1A Knockout by Biallelic and Selection-Free CRISPR Gene Editing in Human Primary Endothelial Cells with Ribonucleoprotein Complexes.

Autor: Brandt CB; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.; Steno Diabetes Center Aarhus, Aarhus University Hospital, 8200 Aarhus, Denmark., Fonager SV; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.; Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark., Haskó J; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark., Helmig RB; Department of Obstetrics and Gynecology, Aarhus University Hospital, 8200 Aarhus, Denmark., Degn S; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark., Bolund L; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.; Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao 266555, China., Jessen N; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.; Steno Diabetes Center Aarhus, Aarhus University Hospital, 8200 Aarhus, Denmark., Lin L; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.; Steno Diabetes Center Aarhus, Aarhus University Hospital, 8200 Aarhus, Denmark., Luo Y; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark.; Steno Diabetes Center Aarhus, Aarhus University Hospital, 8200 Aarhus, Denmark.; Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen, Qingdao 266555, China.
Jazyk: angličtina
Zdroj: Biomolecules [Biomolecules] 2022 Dec 22; Vol. 13 (1). Date of Electronic Publication: 2022 Dec 22.
DOI: 10.3390/biom13010023
Abstrakt: Primary endothelial cells (ECs), especially human umbilical vein endothelial cells (HUVECs), are broadly used in vascular biology. Gene editing of primary endothelial cells is known to be challenging, due to the low DNA transfection efficiency and the limited proliferation capacity of ECs. We report the establishment of a highly efficient and selection-free CRISPR gene editing approach for primary endothelial cells (HUVECs) with ribonucleoprotein (RNP) complex. We first optimized an efficient and cost-effective protocol for messenger RNA (mRNA) delivery into primary HUVECs by nucleofection. Nearly 100% transfection efficiency of HUVECs was achieved with EGFP mRNA. Using this optimized DNA-free approach, we tested RNP-mediated CRISPR gene editing of primary HUVECs with three different gRNAs targeting the HIF1A gene. We achieved highly efficient (98%) and biallelic HIF1A knockout in HUVECs without selection. The effects of HIF1A knockout on ECs' angiogenic characteristics and response to hypoxia were validated by functional assays. Our work provides a simple method for highly efficient gene editing of primary endothelial cells (HUVECs) in studies and manipulations of ECs functions.
Databáze: MEDLINE
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