Fig. 1. CRISPR-DREAM displays potent activation at human promoters, has high specificity and is robust across cell types.
a, dCas9, a gRNA containing two engineered MS2 stem-loops (MS2 SLs) and MCP-fused transcriptional effector proteins are schematically depicted. b, dCas9 and MCP fusion proteins are schematically depicted. Nuclease-inactivating mutations are indicated by yellow bars with dots above. c, The expression levels of dCas9 and dCas9-VP64 (top), FLAG-tagged MCP-mCherry, FLAG-tagged MCP-MSN, FLAG-tagged MCP-p65-HSF1 (middle) and β-tubulin (loading control; bottom) are shown as detected by western blotting in HEK293T cells at 72 h post-transfection. d,e, Relative expression of endogenous human genes after control, DREAM or SAM systems were targeted to their respective promoters using pools of 4 or 3 gRNAs (HBG1 and CD34, respectively; d), or using single gRNAs (ACE2 and HGF, respectively; e), as measured by qPCR. f, RNA-seq data generated after the DREAM or SAM systems were targeted to the HBG1/HBG2 promoter using 4 pooled gRNAs. mRNAs identified as significantly differentially expressed (fold change >2 or <−2 and FDR < 0.05) are shown as red dots. In the top MA plot (CRISPR-DREAM), mRNAs corresponding to HBG1/HBG2 (target genes) are highlighted in light blue. In the bottom MA plot (SAM system), mRNAs corresponding to HBG1/HBG2 (target genes) are highlighted in light gray. mRNAs encoding human components of the MSN or SAM systems shown were also significantly differentially expressed (fold change >2 and FDR < 0.05) and are shown in red. g, Six endogenous genes were activated by DREAM or SAM using a pool of gRNAs (1 gRNA per gene) in HEK293T cells. h,i, OCT4 (h) or HBG1 (i) gene activation by DREAM or SAM systems when corresponding promoters were targeted by 4 gRNAs per promoter in hTERT-MSC or PMBC cells, respectively. All qPCR and RNA-seq samples were processed at 72 h post-transfection. Data are the result of 4 biological replicates for d, e and g and 3 biological replicates for h and i. See the source data for more information. Data are presented as mean ± s.e.m. P values were determined using unpaired two-sided t-test. FDR, false discovery rate.