CRISPRa-activated Genes are Silenced in Differentiated Cells

Researchers from the Institute for Stem Cell and Regenerative Medicine, University of Washington, have taken a significant step forward in applying CRISPR technology within human induced pluripotent stem cells (hiPSCs), aiming to unlock the potential of gene activation during various cellular states.

The study introduced a CRISPR activation (CRISPRa) system into hiPSCs using a deactivated Cas9 (dCas9) fused with activators, targeting the AAVS1 safe harbour site for stable gene expression. Initially successful in activating genes in pluripotent cells, the system faced an unexpected challenge: the expression of the dCas9-activator complex was silenced when cells differentiated into cardiomyocytes and endothelial cells.

To dissect this phenomenon, researchers explored various genomic loci and promoter configurations, only to find that the silencing effect persisted across safe harbour sites and was independent of the promoters used. Interestingly, when dCas9 was replaced with fluorescent protein cDNAs, expression remained stable through differentiation, suggesting a specific response to the CRISPRa components.

Further investigation revealed promoter DNA hypermethylation in silenced lines, hinting at an epigenetic mechanism behind the loss of expression. Treatment with a demethylating agent restored some gene expression, providing a potential workaround and highlighting the need for additional strategies to maintain CRISPRa activity through differentiation.

This study underscores the complexity of applying CRISPR technology in stem cell research. It emphasizes the necessity for thorough pre-application testing to ensure sustained functionality of CRISPRa systems in differentiated cells.

The research was led by Charles Murry and published Monday in Cellular and Molecular Life Sciences. You can read it here.