Figure 2 | Scientific Reports

Figure 2

From: Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen

Figure 2

UPS screen workflow and selection of MYC stability modulators. (A, B) HEK293A cells are engineered to stably overexpress C-terminal MYC sensor and Cas9. A single cell clone (B6) is generated from a multi-clonal cell population, expanded and infected with the CFP-labeled sgRNA encoding lentiviral library at a multiplicity of infection (MOI) of 0.3. After seven or 14 days of infection, puromycin-selected and CFP-positive cells (+ library) were FACS-sorted based on their GFP/mCHERRY ratio (high, low, or unmodified) and compared to uninfected cells (− library). Enrichment of sgRNA in the respective bins is compared to unsorted cell population. (C) sgRNA count on day 7 and day 14 relative to day 0 in cells with high GFP/mCHERRY ratio comparing genes essential for cell survival (lethal) to nonessential genes (NA). (D, E) Plots showing gene enrichment score in high GFP/mCHERRY-sorted cells relative to unsorted cell population visualized as LogFC.

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