Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Abstract

Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.

Figure 1

CRISPR-on activates exogenous transgenes. (A) Schematic of the dCas9VP48-mediated transgene activation in HeLa cells. dCas9VP48 was generated by fusing dCas9 (indicated by black circle) to VP48 domain (indicated by green dimond). sgRNA complementary to rtTA binding site is indicated by small hairpin labeled sgTetO. (B) dCas9VP48 activates TetO::tdTomato transgene in HeLa cells. Upper panel, phase contrast picture of transfected cells; middle panel, tdTomato signal using fluorescent microscopy; bottom panel, FACS analysis of transfected cells. Column i, cells transfected with GFP plasmid; column ii, cells treated with doxycycline; column iii, cells transfected with dCas9VP48 only; column iv, cells transfected with dCas9VP48 and sgTetO. Cells were transfected with the indicated plasmids and 48 h later were analyzed by flow cytometry for tdTomato expression. (C) Schematic of the dCas9VP48-mediated reporter activation in early mouse embryos. dCas9VP48, Nanog::EGFP vector, and 7 sgRNAs targeting Nanog promoter were co-injected into mouse zygotes and cultured into blastocyst stage. (D) dCas9VP48/sgRNA can activate gene in vivo. Left panel, embryos injected with dCas9VP48 and Nanog::EGFP vector; right panel, embryos injected with dCas9VP48, Nanog::EGFP vector and sgRNAs targeting Nanog promoter. Embryos two, three, four days post-injection were shown.

Figure 2

dCas9VP160 activates endogenous genes. (A) Protein architecture of dCas9VP160 compared to VP48. (B) Schematic of the human IL1RN promoter region. Locations of transcription start site (TSS) and start codon (ATG) are indicated. Short lines with number indicate targeting sites of the sgRNAs. (C) Activation of human IL1RN expression in HEK293T cells. Cells were analyzed by qRT-PCR 2 days after transfection with dCas9VP160 and the indicated sgRNAs. (D) Schematic of the human SOX2 promoter region. Locations of TSS and start codon (ATG) are indicated. Short lines with number indicate targeting sites of sgRNAs. (E) Activation of SOX2. Cells were analyzed by qRT-PCR 2 days after transfection with dCas9VP160 and the indicated sgRNAs. (F) Schematic of the human OCT4 promoter region. Locations of transcription start site (TSS) and start codon (ATG) are indicated. Short lines with number indicate targeting sites of sgRNAs. (G) Activation of OCT4. Cells transfected with dCas9VP160 and the indicated sgRNAs were analyzed by qRT-PCR 2 days later. sgTetO-mut, negative control sgRNA. Error bars show SD among triplicates.

Figure 3

Multiple exogenous and endogenous genes were simultaneously activated by CRISPR-on. (A) One exogenous and two endogenous genes were simultaneously activated by CRISPR-on. Cells were analyzed by qRT-PCR 2 days after transfection with dCas9VP160 and the indicated sgRNAs. (B) Three endogenous genes, SOX2, IL1RN and OCT4, can be simultaneously activated by dCas9VP160/sgRNAs. Cells were transfected with dCas9VP160 and the indicated sgRNAs and were analyzed by qRT-PCR 2 days after transfection. The Last three sets of bars represent triple activation experiments using sgSOX2, sgOCT4 and sgIL1RN with three different ratios of sgSOX2:sgIL1RN, keeping the amount of sgOCT4 constant, as indicated by numbers above line. sgTetO-mut, negative control sgRNA. Error bars show SD among triplicates.

Figure 4

Genome-wide expression analysis of cells transfected with the CRISPR-on system. (A) The histogram showing distribution of Log₂ fold changes of gene expression in sample transfected with dCas9VP160/sgTetO over dCas9VP160/sgTetO-mut control. (B) A histogram showing distribution of log₂ fold changes of gene expression in cells transfected with dCas9VP160/sgIL1RN1∼3 over dCas9VP160/sgTetO-mut control. Vertical line marks the fold change of the target gene IL1RN. (C) Column graph showing the Log₂ fold changes of genes upregulated by at least 2-fold in cells transfected with dCas9VP160/sgTetO over dCas9VP160/sgTetO-mut. (D) Column graph showing the Log₂ fold changes of genes upregulated by more than 2-fold in cells transfected with dCas9VP160/sgIL1RN1∼3 over dCas9VP160/sgTetO-mut. Dotted line indicates the 2-fold cut-off.