RNA-guided gene activation by CRISPR-Cas9-based transcription factors

Perez-Pinera et al. (2013). Nat Methods DOI: 10.1038/nmeth.2600 Citations: 1383

Synthetic transcription factors were built from zinc-finger or TALE DNA-binding proteins, which take laborious protein engineering to retarget. This work targeted a dCas9-VP64 transactivator by guide RNA instead and activated nine endogenous human genes. RNA-seq found no off-target activation.

Parts used

dCas9 Catalytically Dead NucleaseVP64FLAG

Key findings

  • A dCas9-VP64 transactivator is retargeted by changing its guide RNA rather than by engineering a new DNA-binding protein. The fusion joins the VP64 acidic transactivation domain to the carboxy terminus of dCas9 (D10A/H840A) and carries an amino-terminal FLAG epitope tag. Guide RNAs give much higher activation of the human IL1RN gene than crRNAs carrying the same four target sequences, and dCas9 expressed without VP64 leaves IL1RN expression unchanged, so the activation domain and the single-guide format are both required. A Surveyor assay confirms that nuclease activity is abolished at the targeted sites.
  • Robust activation by dCas9-VP64 requires several guide RNAs bound at one promoter. Individually transfected guide RNAs against the IL1RN promoter give little activation, while the combination of four acts synergistically. IL-1 receptor antagonist protein is detectable in three of six samples treated with the four-guide combination and in none treated with a single guide RNA or control plasmid.
  • Gene activation by dCas9-VP64 with four guide RNAs is highly specific. By RNA-seq of HEK293T cells, the only transcripts significantly raised relative to control are the four isoforms expressed from the targeted IL1RN locus, at a false discovery rate of 3 × 10^-4 or better. The IL32 gene is moderately downregulated (false discovery rate below 0.03) in cells targeted at either IL1RN or HBG1/2, so the downregulation does not follow from the identity of the activated gene.
  • Targeting four guide RNAs with dCas9-VP64 to the promoters of ASCL1, NANOG, HBG1/2, MYOD1, VEGFA, TERT, IL1B and IL1R2 raises expression of each in HEK293T cells. A single guide RNA suffices at some of these promoters, but the four-guide combination is synergistic at all of them. Increases in Ascl1 and γ-globin protein confirm the mRNA increases, against a low detectable protein baseline in empty-vector controls.
  • Chromatin accessibility does not predict whether dCas9-VP64 will activate a target promoter: DNase-seq accessibility of the nine promoters tested does not correlate with successful activation.
  • dCas9-VP64 activates a target gene in mouse as well as human cells where the guide RNA target sites are conserved. The four guide RNAs against the human ASCL1 promoter also match the mouse promoter and raise ASCL1 mRNA in murine embryonic fibroblasts four days after transfection.
  • dCas9-VP64 activates IL1RN more weakly than TALE-based transcription factors targeted to the same promoter, a trend that also holds at the other targets tested.

Abstract

Technologies for engineering synthetic transcription factors have enabled many advances in medicine and science. In contrast to existing methods based on engineering of new DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Co-expression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.