Multiplex epigenetic editing to activate human pancreatic β-cell genes using different protein domains with CRISPR-dCas9 platform

GIMENEZ CARLA; CURTI LUCIA; HYON, SUNG HO; GROSEMBACHER LUIS; PEREYRA-BONNET FEDERICO

CAPITAL FEDERAL

Congreso; IV International congress in translational medicine; 2018

Universidad de Buenos Aires

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 changing targeted gene expression. Our group showed that CRISPR-on could improve pancreatic cellular reprogramming protocols increasing the expression of lineage-specific transcription factors 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 desing, 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