Recently, a new tool is now available to control gene expression in order to understand gene function and manipulate cell fate. This new tool is called CRIPSR/Cas9, which is a gene-editing tool that employs a genetic system that naturally occurs in bacteria, who use it as a protection against viruses. CRISPR/Cas9 allows scientists to precisely add, remove or replace specific sequences of DNA. It is the most efficient, inexpensive and easiest gene editing tool available to date.
Several laboratories have tried to use CRISPR/Cas9 to activate genes in cells, but such an effort has not always succeeded. However a research team at Hokkaido University’s Institute of Genetic Medicine has developed a powerful new method that uses CRISPR/Cas9 to do exactly that.
In cells, genes have an expression switch called “promoters.” Genes are switched off, or silenced, when their promoters are methylated, which means that islands of C-G bases have a methyl group (a –CH3 group) attached to the cytosine base. The Hokkaido University team wanted to turn an inactivated gene on. The ingeniously combined a DNA repair mechanism, called MMEJ (microhomology-mediated end-joining), with CRISPR/Cas9 to do this. They excised a methylated promoter using CRISPR/Cas9 and then used MMEJ to insert an unmethylated promoter. Thus, they replaced the off-switch signal with an on-switch signal.
The gene that was activated was the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in order to test the efficiency of this technology in cultured cells. Within five days, they found evidence that the genes were robustly expressed. When they activated the OLIG2 gene in cultured human stem cells, the cells differentiated to neurons in seven days with high-efficiency.
Toru Kondo and his colleagues also discovered that their editing tool could be used to activate other silenced promoters. They also found that their system didn’t cause unwanted mutations in other non-target genes in the cells. According to Kondo, this gene editing tool has wide potential to manipulate gene expression, create genetic circuits, or to engineer cell fates.
See Shota Katayama et al., “A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation,” Angewandte Chemie International Edition, 2016; 55(22): 6452 DOI: 10.1002/anie.201601708.