CRISPRa-mediated gene upregulation in mammalian cells.

BACKGROUND

Forkhead box P3 ( ) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the gene (CNS1-3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2).

RESULTS

Using an activator-domain fusion based dCas9 transcription activator, robust upregulation of was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on gene upregulation. Simultaneous targeting of and , a transcription factor known to act in concert with in initiating a Treg phenotype, resulted in upregulation of downstream genes and . When compared to ectopic expression of via plasmid electroporation, upregulation of endogenous via the Cas9-based method resulted in prolonged expression of in Jurkat cells.

CONCLUSIONS

Transfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of promoter can be very potent transcription inducers in comparison to targeting the core promoter. While introduction of genes by conventional methods of gene therapy may involve a risk of insertional mutagenesis due to viral integration into the genome, transient up- or down-regulation of transcription by a CRISPR-dCas9 approach may resolve this safety concern. dCas9-based systems provide great promise in DNA footprint-free phenotype perturbations (perturbation without the risk of DNA damage) to drive development of transcription modulation-based therapies.