Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation

Abstract

SummaryCRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.

Genome-Editing Technologies: Principles and Applications

“…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). Much like zinc fingers and TALEs, methods for achieving conditional gene modulation using dCas9 have also been reported, including the fusion of a dihydrofolate reductase destabilization domain to dCas9, which can provide chemical control over activation, enabling cellular reprogramming or differentiation (Balboa et al 2015). Light-inducible dCas9-based systems capable of providing optical control of gene expression provide another means for achieving conditional control of gene expression (Nihongaki et al 2015;Polstein and Gersbach 2015).…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…However, it remains to be demonstrated that transient transfection of vectors in PSCs can also mediate an increase in protein expression, given the difficulty of achieving the efficient plasmid delivery needed for endogenous gene targeting in difficult-to-transfect human iPSCs and the variability in the induction efficiency. In fact, evidence suggests that an increase in gene expression may be associated with an increase in protein translation [79,160,166]. Although correlation between mRNA abundance and protein synthesis is variable [220], achieving a sizeable -globin gene activation may translate into a higher level of protein expression.…”

“…Tandem repeats of VP16(n) (TAD) such as VP48 (n=3), VP64 (n=4), and VP160 (n=10) are commonly fused to a DNA binding domain in 23 order to target the activator to the gene of interest and promote its transcription [156]. VP96 (n=6) and VP192 (n=12) have been used infrequently [160]. ZFN, TALEN and Cas9 systems attached to such tandem VP16 repeats have been successfully used for transcriptional activation of various endogenous genes [79,161,162].…”

“…They include chemical methods, like cationic liposomes [79], and non-liposome [164] or physical methods, such as electroporation [160]. However, cell viability and transfection efficiency varies with each transfection method [94,217].…”

“…This approach can also be adapted to mediate gene activation by directing epigenetic regulators to the enhancer regions of specific loci [172]. Although variability in the potency of gene activation between different targeted gene regions was observed [160], the activation level was sufficient to enable the replacement of reprogramming factors (OCT4) [160] in iPSC generation or to induce the differentiation of hiPSCs [160,166,215]. For some loci, gene expression levels can be achieved that approach those of native tissue, provided enhanced activation systems are used [166].…”