Fig. 1.

A genome wide CRISPR screen identifies SAFB as a gene required for KRAS4B membrane association. (A) Schematic of the KRAS4B membrane association assay used in the screen (Left). At baseline the Gal4-VP16 transcriptional activator module fused to KRAS4B is sequestered from the nucleus by the native membrane-targeting sequence of KRAS4B but induces expression of GFP upon loss of affinity for membranes. The Right panel shows the various nodes in the trafficking pathway that, upon inhibition (⊣) or enhancement (←), could lead to increased nuclear import of Gal4-VP16-KRAS4B. (B) Results of one of three independent CRISPR screens plotted as read counts for guides targeting each gene in GFP⁺ vs. GFP⁻ cells such that deviation from the diagonal shows enrichment or depletion (Top). Listed on the right are the genes that differed significantly with >100 reads in three of three screens, and on the Bottom is a STRING protein interaction plot showing that all but SAFB are known components of the polyisoprene biosynthetic pathway. (C) U2OS cells edited by CRISPR-Cas9 with either a control (sgNT) or SAFB-targeting guide (sgSAFB) were transfected with either mCherry-KRAS4B alone or mCherry-KRAS4B and GFP-SAFB with a sgSAFB-resistant PAM site and imaged alive with a confocal microscope. (D) U2OS cells in which SAFB or the α subunit of FNTA was silenced with CRISPR-Cas9 or siRNA, as indicated, or treated with a FTI, were transfected with mCherry-KRAS4B and imaged alive as in C (Top). Silencing of SAFB and FNTA was confirmed by immunoblot, shown on the Bottom. In C and D cells representative of >100 per plate are shown. (Scale bar, 20 µm.)