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.

An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression

“…To resolve the question of ERa binding at this site being repressive of IGFBP5-A versus merely upregulating IGFBP5-C, we utilized a transactivator-fused nuclease-defective CRISPR system (13) to activate specific genomic sites in 2q35. We hypothesized that if ERa binding were repressive at this locus under normal conditions, when targeting a definitive transactivator molecule to this site, we would observe the inverse transcriptional response (i.e.…”

Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

“…CRISPR interference (CRISPRi) is a recently developed tool used to study single guide RNA (gRNA)-mediated sequence-specific repression of transcription in both prokaryotic and eukaryotic cells [1][2][3][4][5]. Transcription initiation and elongation of a gene can be interfered by the presence of gRNA:DNA hetero-duplex/dCas9 (a catalytically inactive form of Cas9) complex in its promoter and exons.…”

“…If Krüppel associated box (KRAB) domain is fused to dCas9, repression of target gene is more efficient. Likewise, fusion of a transcription activator such as VP16 (CRISPR-on) can increase target gene expression through three to four gene-specific gRNAs (up to seven) that recognize the proximal promoter of a target gene in cultured cells and in vivo [2]. We applied both CRISPRi and CRISPR-on tools in worm and zebrafish, and demonstrated here that our dCas9 fusion systems modify gene expression at or near their endogenous expression location(s) through target-specific gRNAs (ts-gRNAs).…”

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

“…In eukaryotes, the DSBs are more commonly repaired by the mechanism of error-prone non-homologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletions (indels), around the DSBs . Owing to the simplicity of manipulation and versatility, the CRISPR/Cas9 system has been utilized as an attractive tool for various applications, such as genome-wide screening (Shalem et al, 2014;Zhou et al, 2014), gene repression and activation (Cheng et al, 2013;Doench et al, 2014;Gilbert et al, 2014), targeted fluorescence imaging (Tanenbaum et al, 2014) and novel approaches against pathogens including hepatitis B virus (Lin et al, 2014a;Seeger & Sohn, 2014), human papillomavirus (Kennedy et al, 2014), Epstein-Barr virus (Wang & Quake, 2014;Yuen et al, 2015), malaria (Wagner et al, 2014) and HIV-1 (Ebina et al, 2013;Hu et al, 2014;Ye et al, 2014).…”