CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes

Parts used

Show 10 moreparts→

Key findings

• dCas9 works as a modular platform that delivers any fused effector domain to a locus set only by a coexpressed sgRNA, giving either repression or activation from the same protein scaffold. Mammalian codon-optimized S. pyogenes dCas9 carrying two carboxy-terminal SV40 nuclear localization signals and tagBFP is fused to the KRAB domain of Kox1, the chromo shadow domain of HP1α, the WRPW domain of Hes1, VP64 or the p65 activation domain, and the identity of the fused domain sets whether transcription falls or rises.
• Among repressive chromatin-modifier domains fused to dCas9, only KRAB adds potency. Against an SV40-promoter-driven GFP reporter integrated in HEK293 cells, dCas9-KRAB reduces GFP signal 5-fold while dCas9 alone, dCas9 fused to the HP1α chromo shadow domain, and dCas9 fused to the Hes1 WRPW domain each reduce it only 2-fold.
• Repression by dCas9-KRAB involves no DNA cleavage. The GFP reporter locus retains a wild-type sequence with no detectable indels in knocked-down cells grown for three weeks, so dCas9 silences transcription while remaining nucleolytically inactive.
• Stably expressed dCas9-KRAB silences a reporter gene strongly and durably. Delivered by a minimal lentiviral construct under the spleen focus-forming virus (SFFV) promoter into GFP+ HEK293 cells, it gives substantial knockdown 3 days after sgRNA transfection and strong silencing at 6 days. Six of eight sgRNAs against GFP reduce expression by at least 75%, and the best (NT1) gives about 15-fold repression. The level of remaining GFP varies linearly with the expression level of the sgRNA vector, so repression is tunable by sgRNA dose.
• Repression by dCas9-KRAB is highly specific to the targeted gene. By RNA-seq of GFP+ HEK293 cells expressing dCas9-KRAB with an sgRNA against GFP versus a negative-control sgRNA, GFP is the only significantly suppressed transcript, and averaged across two biological replicates no other gene changes by more than 1.5-fold. The few genes that do shift carry no near match to the sgRNA seed sequence. Expressing either sgRNA alone, or dCas9-KRAB alone, changes little.
• dCas9 and dCas9-KRAB repress endogenous human genes, with the sgRNA target site the dominant variable. Of ten sgRNAs each tiled across 500 bp around the transcription start sites of CD71 and CXCR4 on both template and non-template strands, three per gene give 60% to 80% repression in HeLa cells. Knockdown by dCas9 and by dCas9-KRAB correlates strongly across sgRNAs (R² = 0.89, slope 0.82), so sgRNA binding or local chromatin structure limits repression more than the KRAB domain does, with dCas9-KRAB more repressive on average.
• Two endogenous genes can be silenced at once by transfecting sgRNAs against CD71 and CXCR4 together into HeLa cells stably expressing dCas9-KRAB. Stable lentiviral expression of both the sgRNA and dCas9 or dCas9-KRAB keeps CD71 and CXCR4 knocked down at 7 to 8 days, so CRISPRi can replace RNAi for sustained suppression of endogenous genes.
• Targeting dCas9-KRAB to promoter-proximal cis-acting elements silences the gene they control. Against six sgRNAs tiled across the SV40 early promoter of a GFP reporter, dCas9 alone has no effect on transcription while dCas9-KRAB represses GFP efficiently, so the KRAB domain is what matters at promoter-proximal elements even though dCas9 alone can repress elsewhere.
• dCas9 represses transcription in the budding yeast Saccharomyces cerevisiae, and a fused repressor domain deepens it. Against a TEF1-GFP fusion expressed from its endogenous locus, dCas9 alone gives 18-fold repression and dCas9 fused to the mammalian Mxi1 repressor domain gives 53-fold. Repression in yeast is therefore several-fold stronger than the 5- to 15-fold seen in human cells.
• dCas9 can block a transcription factor from reaching its binding sites. In S. cerevisiae carrying a TetON-Venus reporter and the rtTA activator, rtTA strongly activates the reporter, and targeting dCas9 to the tetO sites blocks that induction with 115-fold repression.
• A dCas9 fusion can activate as well as repress transcription from the same scaffold: both dCas9-VP64 and dCas9 fused to the p65 activation domain activate a Gal4 UAS-GFP reporter in HEK293 cells when targeted by an sgRNA against the Gal4 upstream activation sequence.

Abstract

The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.