CRISPR RNA-guided activation of endogenous human genes

Abstract

Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.

Figure 1. RNA-guided activation of endogenous human genes

(a) Schematic depicting recruitment of dCas9-VP64 fusion protein to a specific genomic target sequence by an sgRNA. (b) 16 sgRNAs targeted to the endogenous human VEGFA gene promoter. Red arrows represent the first 20 nts of the sgRNA pointing 5’ to 3’. Grey bars indicate DNaseI hypersensitive sites previously defined in human 293 cells, numbered relative to the transcription start site (black arrow). (c) Activation of VEGFA protein expression in 293 cells by various sgRNAs, each expressed with (blue bars) or without (red bars) dCas9-VP64. Asterisks indicate samples that are significantly elevated above the off-target control as determined by a paired, one-sided t-test (n = 3, P < 0.05); error bars represent s.e.m. (d) Six sgRNAs targeted to the endogenous human NTF3 gene promoter represented as in (b) Grey line indicates region of potential open chromatin identified from the ENCODE DNaseI hypersensitivity track on the UCSC genome browser with the thicker part of the bar indicating the first transcribed exon. Numbering shown is relative to the transcription start site (black arrow). (e) Activation of NTF3 gene expression by RNA-guided dCas9-VP64 in 293 cells. Relative expression of NTF3 mRNA is shown for 293 cells co-transfected with the indicated amounts of codon-optimized dCas9-VP64 and NTF3 -targeted sgRNA expression plasmids. Asterisks indicate samples that are significantly greater than the off-target sgRNA control as determined by a paired, one-sided t-test (n = 3, P < 0.05); error bars represent s.e.m.

Figure 2. Synergistic activation of the endogenous human VEGFA and NTF3 genes by RNA-guided activators

(a) Multiplex sgRNA expression induces synergistic activation of VEGFA protein expression by dCas9-VP64 protein. The calculated sum of mean VEGFA protein expression induced by individual sgRNAs is shown for each combination as green bars. Asterisks indicate all combinations that were found to be significantly greater than the expected sum as determined by an analysis of variance (ANOVA) (n = 3, P < 0.05). Error bars represent standard errors of the mean. (b) Multiplex sgRNA expression induces increased activation of NTF3 mRNA expression by dCas9-VP64 protein. Relative expression of NTF3 mRNA is shown for 293 cells in which the indicated individual or combinations of sgRNAs were co-expressed with dCas9-VP64. The calculated sum of mean NTF3 mRNA expression induced by individual sgRNAs is shown for each combination as green bars. P values were determined by a paired, one-sided T-test (n = 3). Error bars represent standard errors of the mean.