Figure 5. dCas9-SunTag allows genetic rewiring of cells through activation of endogenous genes.

(A) Schematic of gene activation by dCas9-VP64 and dCas9-SunTag-VP64. dCas9 binds to a gene promoter through its sequence specific sgRNA (red line). Direct fusion of VP64 to dCas9 (top) results in a single VP64 domain at the promoter which poorly activates transcription of the downstream gene. In contrast, recruitment of many VP64 domains using the SunTag potently activates transcription of the gene (bottom). (B–D) K562 cells stably expressing dCas9-VP64 or dCas9-SunTag_10x-VP64 were infected with lentiviral particles encoding indicated sgRNAs, as well as BFP and a puromycin resistance gene and selected with 0.7 µg/ml puromycin for 3 days to kill uninfected cells. (B–C) Cells were stained for CXCR4 using a directly labeled α-CXCR4 antibody and fluorescence was analyzed by FACS. (D) Trans-well migration assays (see experimental procedures) were performed with indicated sgRNAs. Results are displayed as the fold change in directional migrating cells over control cell migration. (E) dCas9-VP64 or dCas9-SunTag_10x-VP64 induced transcription of CDKN1B with several sgRNAs. mRNA levels were quantified by qPCR. (F) Doubling time of control cells or cells expressing indicated sgRNAs was determined (See Experimental Procedures section). Graphs in (C, D and F) are averages of three independent experiments. Graph in (E) is average of two biological replicates, each with two or three technical replicates. All error bars indicated standard error of the mean (SEM). See also figure S4.