Selection-free endogenous tagging of cell lines by bicistronic co-expression of the surface antigen NGFR

Abstract

Endogenous protein tagging, in contrast to exogenous overexpression of tagged proteins, allows to characterize specific protein functions under defined physiological or pathophysiological conditions without the influence of non-physiological protein levels. The development of generic and homology-independent tagging strategies, exploiting the CRISPR/spCas9 gene editing system in combination with generic tag donor plasmids, allows targeted and precise gene modification in mammalian cells for almost any desirable gene.So far, fluorescent tags or antibiotic resistance cassettes coupled to the endogenous fusion protein expression have been applied to isolate correctly modified clones. However, both can be challenging, especially when endogenously controlled expression of the tagged protein is weak or regulated by cellular signals. Here, we expand the strategy to selection-free endogenous tagging by exploiting exogenous co-expression of surface antigens. These endogenously regulated, but still easily accessible surface antigens allow simple identification and isolation of clones harboring correctly tagged alleles via common sorting procedures (e.g. FACS/MACS).Using metabolically controlled interaction studies of the endogenously tagged mTORC1-regulating GATOR2 complex protein WDR59, we show that endogenous GFP-labeling does not affect complex association of fusion proteins and downstream signaling via mTORC1. In addition, exogenous co-expression of the NGFR surface antigen does not influence conditional protein-protein interactions. • A method for selection-free, site-specific, homology-independent endogenous genetic tagging. • Production of fusion genes for protein visualization in living cells or determination of protein-protein-interactions. • Expression of a fusion protein mirroring physiological expression in its natural genetic context.

Keywords

• Selection-free endogenous tagging of cell lines