Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

Abstract

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

A potent Cas9-derived gene activator for plant and mammalian cells

“…Furthermore, since TAL is known as an active TAD in yeast 20 , we speculated that the dCas9-TV-mediated transcriptional activation may also work for mammalian cells, which are distantly related to Arabidopsis and rice cells. Indeed, when we targeted ASCL1 or OCT4 in human embryonic kidney (HEK) 293T cells with a single sgRNA 2,23 , we detected induction of endogenous ASCL1 by 46-fold and OCT4 by 14.6-fold, whereas dCas9-VP64 failed to enhance the expression of either gene (Fig. 2f).…”

Genome-Editing Technologies: Principles and Applications

“…Effector domains are fused to the carboxyl terminus of the dCas9 protein and can modulate gene expression from either strand of the targeted DNA sequence (Farzadfard et al 2013;Maeder et al 2013a;Perez-Pinera et al 2013). Genome-scale activation studies have indicated that the most robust levels of activation are generally observed when dCas9 activators are targeted to -400 to -50 bp upstream from the transcriptional start site (Gilbert et al 2014;Hu et al 2014). Additionally, dCas9 can inhibit gene expression by simply blocking transcriptional initiation or elongation through a process known as CRISPR interference (Qi et al 2013), although fusing dCas9 to transcriptional repressor domains can also lead to efficient silencing from the promoter Zalatan et al 2015).…”

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities

“…For example, gene activation is stronger when dCas9 fusions to transactivators are co-expressed with multiple gRNAs targeting the same promoter [46–50]. However, using multiple gRNAs may also increase the off-target binding activity of dCas9 yielding confounding results.…”