CRISPR activation

Unlike traditional CRISPR-Cas9, which introduces double-strand breaks to edit genes, CRISPRa employs a modified, catalytically inactive Cas9 (dCas9) fused with transcriptional activators to target promoter or enhancer regions, thereby boosting gene transcription.

Such systems are usable for many purposes including but not limited to, genetic screens and overexpression of proteins of interest.

Similar to its unmutated form, dCas9 is used in CRISPR systems along with gRNAs to target specific genes or nucleotides complementary to the gRNA with PAM sequences that allow Cas9 to bind.

The point mutations D10A and H840A change 2 important residues for endonuclease activity that ultimately results in its deactivation.

The activation domain can recruit general transcription factors or RNA polymerase to the gene sequence.

Activation domains can also function by facilitating transcription by stalled RNA polymerases, and in eukaryotes can act to move nucleosomes on the DNA or modify histones to increase gene expression.

The use of three transcription factors, as opposed to solely Vp64, results in increased expression of targeted genes.

Utilizing MS2, p65, and HSF1 proteins, dCas9-SAM system recruits various transcriptional factors working synergistically to activate the gene of interest.In order to assemble different transcriptional activators, the dCas9-SAM system uses a modified single guide RNA (sgRNA) that has binding sites for the MS2 protein.

The antibodies successfully bound to SunTag polypeptides and activated target CXCR4 gene in K562 cell lines.

[11] Comparing with the dCas9-VP64 activation complex, they were able to increase the CXCR4 gene expression 5-25 times greater in K562 cell lines.

Removing the light from the cell results in only dCas9 remaining at the target gene, so expression is not increased.

In the presence of the chemical RAP, an FRB domain fused to a chromatin modifying complex binds to FKBP.

Whenever RAP is added to the cells, a specific chromatin modifier complex can be targeted to the gene.

[15] The versatility of sgRNAs allows dCas9 activators to increase the expression of any gene within an organism's genome.

A paper demonstrated that genome wide activation could be used to determine which proteins are involved in mediated resistance to a specific drug.

For one of the two genes tested, the dCas9 activator changes the number and size of leaves and made the plants better able to handle drought.

The authors conclude that the dCas9 activator can create phenotypes in plants that are similar to those observed when a transgene is inserted for overexpression.

Scientists used the targeting and increased expression of several genes to examine the processes involved in regeneration and carcinomas of the liver.

Complementary base pairing between the sgRNA and genomic DNA allows targeting of Cas9 or dCas9
Complementary base pairing between the sgRNA and genomic DNA allows targeting of Cas9 or dCas9
The dCas9-VPR activator increases transcription at the gene that it targets.
The dCas-SAM system uses msgRNA that has attached aptamers for different transcriptional factors (MS2,p65 and HSF1) to bind.
The use of the Suntag system allows multiple antibodies fused to VP64 to bind to dCas9-Suntag. That in turn recruits RNA polymerase and increases gene expression.