Establishing a HEK293A Cell Line Stably Expressing hMSR1 and a Membrane-Anchored Fluorescent Protein via CRISPR-Cas9-Mediated AAVS1 Safe Harbor Knock-in
HUANG Yabing1,2, LI Zhenhang1,2, CHI Mingwei3, BEI Zhuchun2*, XU Qin1*, SONG Yabin2*
This study aimed to enhance the adhesion and label the plasma membrane of HEK293A cell line by knocking in the hMSR1 (human macrophage scavenger receptor 1) and membrane-anchored fluorescent protein genes at a specified site. Using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing technology, the hMSR1 and membrane-anchored fluorescent protein bicistronic expression cassette was precisely integrated into the AAVS1 (adeno-associated virus integration site 1). Cell adherence was determined by crystal violet staining following PBS washing; cell proliferation was measured using CCK-8 assay; transfection efficiency and response to signaling pathway activation in cells were assessed by luciferase reporter assays; and fluorescent protein localization was analyzed by fluorescence microscopy. Four recombinants with correct integration of the target genes were selected, all of which showed significantly stronger adherence to tissue culture plastic than wild-type HEK293A cells and correct membrane localization of the fluorescent protein, with no significant changes in proliferation profile or loss of responsive to basic signaling pathway activation.
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