The CRISPR based tool kit is evergrowing and is becoming more and more advanced daily. In this article, we will look into transcriptional regulation with CRISPR and how it has progressed over time. Transcriptional regulation is whereby the gene expression of a certain gene can either be increased or decreased. This can be useful to determine the function of a certain gene and it could have therapeutic potential. Transcriptional regulation uses dCas which is dead Cas, therefore it has no DNA cutting abilities. This technique is highly specific as the dCas is guided to the specific gene with guide RNA.
First Generation Tools
It was discovered that dCas9 was able to inhibit gene expression of a specific gene by literally blocking the RNA polymerase from binding to the gene promoter/moving down the sequence. The ability of dCas9 to inhibit gene expression was coined as CRISPR interference (CRISPRi). Various activators and inhibitors were then fused to dCas to see whether this could impact transcriptional regulation. It was observed that dCas9 fused with KRAB was able to repress gene expression consistently and an activator like VP16/VP64 or Kappa B fused to dCas9 was able to increase gene expression levels of a certain gene, this was coined as CRISPR activation (CRISPRa). With the conventional gene editing, you can get a complete gene knockout but with transcriptional regulation, the effect of various gene expression levels on the cell function can be determined.
Second Generation Tools
To make second-gen transcription regulation tools more efficient, multiple activators were fused onto dCas9 to enhance the gene expression. There are a few examples below which show this:
SunTag - Tanenbaum and colleagues developed a system called dCas9-SunTag. This is whereby dCas9 is fused to SunTag which is a peptide array capable of binding to an scFv (single-chain variable fragment) bound to VP64, in other words, the system is capable of recruiting several activators.
VPR - The VPR system uses dCas9–VPR which is dCas9 fused to three activators consisting of VP64, p65AD, and Rta. Instead of using one activator, three are used which results in enhanced gene expression.
Synergistic Activation Mediator (SAM) - The SAM system uses dCas9–VP64 but with a modified guide RNA that contains a binding site for MS2 binding protein (MCP) which is fused to various activators such as p65 and HSF1 that can enhance gene expression.
There are many more variations of these systems but these are the main ones.
Third Generation Tools
Ligand-inducible control of gene expression comes under the third generation tools, it allows transcriptional regulation to become a more controlled technique. The dCas can have a domain added that can be induced by a specific drug or optically, therefore, the addition of a certain drug or light of a certain wavelength can cause changes that result in the recruitment of various effectors (transcriptional activators or inhibitors) and then activation of the CRISPR-Cas system.
An example of this is using the drug rapamycin to induce CRISPR based transcriptional activation.