Fig. 3: A fluorescent reporter measures endogenous PSMB5 expression in live cells. | Nature Communications

Fig. 3: A fluorescent reporter measures endogenous PSMB5 expression in live cells.

From: Loss-of-function mutations in the dystonia gene THAP1 impair proteasome function by inhibiting PSMB5 expression

Fig. 3

A–C CRISPR/Cas9-mediated knock-in of GFP into the endogenous PSMB5 locus. A schematic representation of the procedure is shown in (A). Transfection of HEK-293T cells with Cas9, an sgRNA targeting PSMB5 and a donor template resulted in ~10% GFP-positive cells (B), which were purified by FACS and single cell cloned (C). D–G THAP1 ablation reduces PSMB5 expression. CRISPR/Cas9-mediated disruption of THAP1 in a PPSMB5-GFP reporter clone (expressing exogenous PSMB5 to ensure viability) reduced PPSMB5-GFP expression (D), permitting the derivation of GFPdim THAP1 KO clones (E). These THAP1 KO clones exhibited greatly reduced expression of PSMB5 by qRT-PCR (using primers annealing to the 3’UTR to ensure selective amplification of the endogenous transcripts) (F), and an absence of THAP1 protein by immunoblot (a non-specific band is indicated by an asterisk) (G). Data in (F) represent mean values of n = 3 technical replicates ± s.d. Source data are provided as a Source Data file.

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