CRISPR activation
CRISPR activation is a gene regulation technique that utilizes an engineered form of the CRISPR-Cas9 system to enhance the expression of specific genes without altering the underlying DNA sequence. Unlike traditional CRISPR-Cas9, which introduces double-strand breaks to edit genes, CRISPRa employs a modified, catalytically inactive Cas9 fused with transcriptional activators to target promoter or enhancer regions, thereby boosting gene transcription. This method allows for precise control of gene expression, making it a valuable tool for studying gene function, creating gene regulatory networks, and developing potential therapeutic interventions for a variety of diseases.
Like for CRISPR interference, the CRISPR effector is guided to the target by a complementary guide RNA. However, CRISPR activation systems are fused to transcriptional activators to increase expression of genes of interest. Such systems are usable for many purposes including but not limited to, genetic screens and overexpression of proteins of interest.
The most commonly used effector is based on Cas9, but other effectors like Cas12a have been used as well.
Components
dCas9
Cas9 Endonuclease Dead, also known as dead Cas9 or dCas9, is a mutant form of Cas9 whose endonuclease activity is removed through point mutations in its endonuclease domains. 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. Cas9 ordinarily has 2 endonuclease domains called the RuvC and HNH domains. The point mutations D10A and H840A change 2 important residues for endonuclease activity that ultimately results in its deactivation. Although dCas9 lacks endonuclease activity, it is still capable of binding to its guide RNA and the DNA strand that is being targeted because such binding is managed by other domains. This alone is often enough to attenuate if not outright block transcription of the targeted gene if the gRNA positions dCas9 in a way that prevents transcriptional factors and RNA polymerase from accessing the DNA. However, this ability to bind DNA can also be exploited for activation since dCas9 has modifiable regions, typically the N and C terminus of the protein, that can be used to attach transcriptional activators.Guide RNA
See: Guide RNA, CRISPRA small guide RNA, or gRNA is an RNA with around 20 nucleotides used to direct Cas9 or dCas9 to their targets. gRNAs contain two major regions of importance for CRISPR systems: the scaffold and spacer regions. The spacer region has nucleotides that are complementary to those found on the target genes, often in the promoter region. The scaffold region is responsible for formation of a complex with Cas9. Together, they bind Cas9 and direct it to the gene of interest. Since the spacer region of a gRNA can be modified for any potential sequence, they give CRISPR systems much more flexibility as any genes and nucleotides with a sequence complementary to the spacer region can become possible targets.
Transcriptional activators
See: Transcriptional Activator, Transcription FactorTranscriptional Activators are protein domains or whole proteins linked to dCas9 or sgRNAs that assist in the recruitment of important co-factors as well as RNA Polymerase for transcription of the gene targeted by the system. In order for a protein to be made from the gene that encodes it, RNA polymerase must make RNA from the DNA template of the gene during a process called transcription. Transcriptional activators have a DNA binding domain and a domain for activation of transcription. 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. These activators can be introduced into the system through attachment to dCas9 or to the sgRNA. Some researchers have noted that the extent of transcriptional upregulation can be modulated by using multiple sites for activator attachment in one experiment and by using different variations and combinations of activators at once in a given experiment or sample.
Expression system
An expression system is required for the introduction of the gRNAs and Cas9 proteins into the cells of interest. Typically employed options include but are not limited to plasmids and viral vectors such as adeno-associated virus vector or lentivirus vector.Specific activation systems
VP64-p65-Rta
The VP64-p65-Rta, or VPR, dCas9 activator was created by modifying an existing dCas9 activator, in which a Vp64 transcriptional activator is joined to the C terminus of dCas9. In the dCas9-VPR protein, the transcription factors p65 and Rta are added to the C terminus of dCas9-Vp64. Therefore, all three transcription factors are targeted to the same gene. The use of three transcription factors, as opposed to solely Vp64, results in increased expression of targeted genes. When different genes were targeted by dCas9, they all showed significantly greater expression with dCas9-VPR than with dCas9-VP64. It has also been demonstrated that dCas9-VPR can be used to increase expression of multiple genes within the same cell by putting multiple sgRNAs into the same cell.dCas9-VPR has been used to activate the neurogenin 2 and neurogenic differentiation 1 genes, resulting in differentiation of induced pluripotent stem cells into induced neurons. A study comparing dCas9 activators found that the VPR, SAM, and Suntag activators worked best with dCas9 to increase gene expression in a variety of fruit fly, mouse, and human cell types.
Synergistic activation mediator
To overcome the limitation of the dCas9-VP64 gene activation system, the dCas9-SAM system was developed to incorporate multiple transcriptional factors. 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 that has binding sites for the MS2 protein. Hairpin aptamers are attached to the tetra loop and the stem loop 2 of the sgRNA to become binding sites for dimerized MS2 bacteriophage coat proteins. As the hairpins are exposed outside of the dCas9-sgRNA complex, other transcriptional factors can bind to the MS2 protein without disrupting the dCas9-sgRNA complex. Thus, the MS2 protein is engineered to include p65 and HSF1 proteins. The MS2-p65-HSF1 fusion protein interacts with the dCas9-VP64 to recruit more transcriptional factors onto the promoter of the target genes.
Using the dCas9-SAM system, Konermann et al. identified genes in melanoma cells that confer resistance to a BRAF inhibitor. In another study, Zhang et al. employed the dCas-SAM system in human cell lines to reactivate latent HIV provirus, triggering apoptosis of infected cells.
SunTag
The SunTag activator system uses the dCas9 protein, which is modified to be linked with the SunTag. The SunTag is a repeating polypeptide array that can recruit multiple copies of antibodies. Through attaching transcriptional factors on the antibodies, the SunTag dCas9 activating complex amplifies its recruitment of transcriptional factors. In order to guide the dCas9 protein to its target gene, the dCas9 SunTag system uses sgRNA.Tanenbaum et al. are credited for creating the dCas9 SunTag system. For the antibodies, they employed GCN4 antibodies which was bound to transcriptional factor VP64. In order to transport the antibodies to the nuclei of the cells, they attached NLS tag. To confirm the nuclear localization of the antibodies, sfGFP was used for visualization purpose. Therefore, the GCN4-sfGFP-NLS-VP64 protein was developed to be interact with dCas SunTag system. The antibodies successfully bound to SunTag polypeptides and activated target CXCR4 gene in K562 cell lines. Comparing with the dCas9-VP64 activation complex, they were able to increase the CXCR4 gene expression 5-25 times greater in K562 cell lines. Not only was there a greater CXCR4 protein overexpression but also CXCR4 proteins were active to further travel on the transwell migration assay. Thus, the dCas9-SunTag system can be used to activate genes that are present latently such as virus genes.