CRISPR RNA-guided activation of endogenous human genes

Maeder et al. (2013). Nat Methods DOI: 10.1038/nmeth.2598 Citations: 1301

dCas9 had been used to repress genes and to activate reporters, but not to activate endogenous human genes. This work fused VP64 to dCas9 and raised VEGFA and NTF3 expression with single and multiple sgRNAs. Several sgRNAs at one promoter act synergistically.

Parts used

dCas9 Catalytically Dead Nuclease VP64 U6 Promoter CMV Promoter 3xFLAG EGFP

Key findings

A dCas9-VP64 fusion activates endogenous human genes when targeted by sgRNAs, where dCas9 alone does not. The fusion joins the synthetic VP64 activation domain to the carboxy terminus of dCas9 (D10A/H840A) through a Gly4Ser linker. Activation requires the VP64 domain: sgRNAs expressed without the fusion, dCas9-VP64 without an sgRNA, and dCas9-VP64 with an sgRNA against an unrelated EGFP reporter sequence all leave target expression unchanged.
Of 16 sgRNAs tiled across three DNase I hypersensitive sites upstream of, downstream of, and at the VEGFA transcription start site, 15 raise VEGFA protein in human 293 cells when coexpressed with dCas9-VP64. All 6 sgRNAs targeting a predicted DNase hypersensitive site in the human NTF3 promoter raise NTF3 transcript levels, with mean activation varying over a four-fold range between sgRNAs. Across both genes 21 of 22 sgRNAs tested give significant activation, so most sgRNAs at an accessible promoter region work, but their potency varies substantially even at the same locus.
Activation by dCas9-VP64 is dose dependent: reducing the amount of transfected sgRNA and dCas9-VP64 expression plasmid reduces NTF3 activation.
Multiple sgRNAs targeting one promoter act synergistically with dCas9-VP64, producing expression above the sum of the individual sgRNAs. Four sgRNAs against VEGFA (V1 to V4), and subsets of three of them, synergistically raise VEGFA protein. Three sgRNAs against NTF3 (N4, N5, N6) and all pairs of them raise NTF3 expression, with three of the four combinations exceeding the expected additive effect. Because synergy requires several activators bound at one promoter, multiple sgRNA and dCas9-VP64 complexes can occupy the same promoter and function together in a single cell.
Codon optimizing dCas9-VP64 does not increase the VEGFA activation it achieves over the non-optimized fusion.
dCas9-VP64 activates VEGFA and NTF3 less strongly than VP64-based TALE activators targeted to the same endogenous genes, so an RNA-guided activator is easier to retarget than a TALE activator but not more potent at these loci.

Abstract

Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.