Image by CellPress: Development and Applications of CRISPR-Cas9 for Genome Engineering
iGEM Project | Part 9
After exposing the molecular mechanism behind Cas9 activity in the previous article, let us introduce you dCas9 and its valuable applications.
Cas9 has two domains (sites of a protein with a specific catalytic or structural function) that can “cut” target DNA. What happens when such cutting domains are mutated? You get a “dead” Cas9 (dCas9), a protein that lacks catalytic activity but keeps its characteristic as an RNA-driven system for specific DNA targets. dCas9 is no useful for genome editing but can be taken advantage of for several other purposes.
As an example, dCas9 gene can be fused with GFP or another fluorescent protein, by linking its coding DNA sequence to the protein of interest gene, to monitor changes in the organisation of chromatin in living cells.
Moreover, the CRISPR/dCas system can be used to modify DNA expression by changing chromatin conformation through fusion with proteins that naturally play this role.
Have you thought of another possible application yet? We can still talk about CRISPR interference (CRISPRi)! dCas9 can stop the expression of a gene just by binding its DNA sequence and occupying the space needed for other proteins to carry its transcription out. CRISPRi effects are remarkable in bacteria.
There are many CRISPRi studies in scientific literature, most of which are ongoing and promising. Much of this research focuses on counteracting bacterial infections in humans, animals and cultivated plants: by designing an appropriate guide RNA, it is possible to target essential genes for the regulation of bacterial metabolic pathways or antibiotic resistance genes.
Additionally, there is another kind of CRISPR tool: gene activation by CRISPR! CRISPRa methods can increase the expression of a target gene, leading to its overexpression.
Okay, we’re done by now… but biotechnologies and scientists certainly are not!