Epigenetic marks such as DNA methylation or histone acetylation play an important role in gene regulation. Catalytically inactive CRISPR-associated 9 nuclease (dCas9) (CRISPR-dCas9) can be directed by short guide RNAs (gRNAs) to modifying DNA methylation or histone acetylation to change targeted gene expression. Our group showed that CRISPR-on could improve pancreatic cellular reprogramming protocols increasing the expression of lineage-specific transcription factors (TF) and genes. However, the potency and effectiveness of different CRISPR-dCas9 platform and its convenient use to maximize the efficiency/cost ratio, in pancreatic genes remain unexplored. In this work we used three diferentes CRISPR-on platform: CRISPR/dCas9-VP160 (transcription activation, Addgene#48226), dCas9-TET1 (DNA demethylation, #82559) and dCas9P300 (histone acethylation, #61357), to activate human pancreatic TF/genes in HEK293 cells. Four to five guides (sgRNAs) per gene were designed, cloned in sgRNA vector (#47108) and co-transfected with the corresponding CRISPR-dCas9 platform. All gene expression levels were measured by qPCR in 2-4 biological replicates. dCas9-VP160 and dCas9-P300 platform showed activation in PDX1, NEUROG3, PAX4 and INS, being dCas9-P300 the only group with significant
results compared to the control (p<0.005). In our hands dCas9-TET1 did not turn most of these genes on, despite observing partial demethylation in INS promoter by 5hmC analysis. Interestingly, when we used the three platforms together, we observed a significant decrease in gene activation (p<0.005). In addition, we evaluated the individual gRNA activation efficiency in dCas9-P300 and dCas9- VP160 platform. dCas9-VP160 needed several sgRNAs per gene to have a significant activation, while dCas9-P300 showed that using one guide can be sufficient to generated a significant gene activation (p>0.005). In addition, we showed for the first time that an additive effect is possible for dCas9-P300 when the most efficient sgRNAs are combined (p<0.005).These results allowed us to reduce the RNA guides from 19 to 5 and to employ multiplex-dCas9-P300 gene activation in the most efficient way. Next, we compared lentiviral dCas9-P300 vs episomal dCas9-P300 plasmid deliveries. For this, we generated HEK293-Lentivial-dCas9- P300 transgenic cell line and used synthetic RNA guides. As a result we demonstrated similar PDX1, NEUROG3, PAX4 and INS activation efficiency, with both lentiviral and plasmid vectors. Finally, we activated pancreatic genes in human induced pluripotent stem cells (hiPSc) generating and using a hIPS-LentiviraldCas9- P300 transgenic cell line with synthetic RNA guides. In conclusion, this work provides a basic knowledge of different CRISPR-dCas9 platform, giving new clues in terms of mechanisms, sgRNA amount and delivery systems, to manipulated pancreatic human gene regulation, providing a proof-of-principle for future CRISPRTherapeutics.