Figure 1
Design and assembly of the UPS screen. (A) The UPS gene cassette drives the expression of a single transcript containing the GFP-fusion to the gene of interest (GOI) and mCHERRY, separated by the chysel peptide (CHY). The sgRNA library targeting the proteasomal pathway components is introduced into mammalian cells that stably express the UPS cassette and Cas9 through lentiviral infection. Stability modulation through sgRNA-mediated knockout is represented by changes in GFP/mCHERRY signal ratio and FACS-sorted into separate bins, followed by NGS sequencing. (B) Confocal microscopy of HEK293A cells expressing either Mock DNA, GFP-chysel-mCHERRY, or N- or C-terminal GFP-CHYSEL-mCHERRY tag to MYC ORF. (C) Single cell clone B6 was treated with MLN4942 ([1 µM]) and Bortezomib ([50 nM]) for 24 h and GFP/mCHERRY ratio was compared to DMSO control using FACS based analysis as well as western blot. The Y-axis scaling (Normalized To Mode) is a feature in the FACS analysis program flowjo based on normalizing to the peak height at mode of the distribution (the maximum Y-axis value in the absolute count = 100%).(D) Cycloheximide chase assay to compare the half-life of wild type MYC and MYC-GFP-chysel-mCHERRY in HEK293A.
