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

Abstract

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression 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.

Figure 1

RNA-guided activation of the human IL1RN gene by dCas9-VP64. (a) An RNA-guided transcriptional activator was created by fusing dCas9 (D10A/H840A) to the VP64 transactivation domain. dCas9-VP64 recognizes genomic target sites through the hybridization of a guide RNA (gRNA) to a 20 bp target sequence. (b) The structure of dCas9-VP64 is shown, including Flag and HA epitope tags, two nuclear localization signals (NLS), and the positions of the RuvC- and HNH-like endonuclease domains that target the noncomplementary and complementary DNA strands, respectively, if not inactivated by the mutations D10A and H840A.(c) Expression plasmids for four gRNAs or crRNA/tracrRNAs targeted to sequences in the IL1RN promoter were co-transfected with the dCas9-VP64 expression plasmid into HEK293T cells. Activation of IL1RN expression was assessed by qRT-PCR. (d) The four gRNA expression plasmids were co-transfected with dCas9-VP64 individually or in combination. Robust gene activation was observed by qRT-PCR only in response to the combination of gRNAs. (e) Activation of IL1RN expression was confirmed by assessing secretion of the IL-1ra gene product into the media by ELISA. IL-1ra was only detectable above background in three of the six samples treated with the combination of gRNAs. For (c-e), data are shown as the mean ± s.e.m (n = 3 independent experiments). Treatment with the combination of gRNAs was statistically different than all other treatments (*P ≤ 0.05) by Tukey’s test. (f) RNA-seq was performed on samples treated with empty expression vector (n = 2) or co-transfected with the expression plasmids for dCas9-VP64 and the four gRNAs targeting IL1RN (n = 2). The only statistically significant changes in gene expression between these treatments were an increase in the four IL1RN isoforms (false discovery rate ≤ 3 × 10⁻⁴) and a decrease in IL32 (false discovery rate = 0.03).

Figure 2

RNA-guided activation of human genes relevant to cell and gene therapy, genetic reprogramming, and regenerative medicine. HEK293T cells were transfected with the dCas9-VP64 expression plasmid and four gRNAs individually or in combination. Target gene expression was measured by qRT-PCR and normalized to GAPDH mRNA levels. Data are shown as the mean ± s.e.m (n = 3 independent experiments). Treatment with the combination of gRNAs was statistically different than all other treatments (*P < 0.05) by Tukey’s test.