CRISPR-mediated Gene activation or CRISPRa (Part 51- CRISPR in gene editing and beyond)
Welcome to the 51st part of the multi-part series on applications of CRISPR in gene editing and beyond.
Although the CRISPR-Cas technique has revolutionized gene editing, scientists are always looking for new possibilities that the CRISPR-Cas system can do? CRISPR-Cas system has the potential to reversibly activate or silence genes with the help of the dead Cas9 enzyme, simply referred to as dCas9. We have already discussed in our previous chapters that dead Cas9 is a mutant in which both cleavage domains HNH and RuVC of Cas9 are inactivated. Although dCas9 can no longer cleave DNA, it can still bind target DNA with the same precision when guided by sgRNA.
When transcriptional activators are fused to dCas9, the resulting complex can activate the expression of the desired gene. Gene activation by dCas9 is called CRISPR activation or simply written as CRISPRa.
Transcriptional Activators are the proteins that bind to the enhancers or promoter-proximal elements. After binding, they assist in the recruitment of transcription machinery, including transcriptional co-factors and RNA Polymerase, to the promoter for transcription of the desired gene, i.e., making RNA from the DNA template of the gene.
Transcriptional activators consist of a DNA binding domain and a transcription activation domain. Through the activation domain, additional transcription factors or RNA polymerase can be recruited to the promoter site, thereby triggering the transcription of the targeted gene.
For activation of gene expression in eukaryotic cells, the transcriptional activators used are VP64 or p65. VP64 is a synthetic tetramer of the activation domain of Herpes Simplex Viral Protein 16, which is a potent transcriptional activator that can activate the transcription of viral genes required for viral replication. On the other hand, the p65 is a subunit of the NF-kB transcription factor and is involved in many cellular processes, including inflammation and immune response.
When these transcriptional activators are attached to dCas9, they can activate the target gene expression. This approach can cause moderate activation of the gene by 2–5 fold.
CRISPR activation (CRISPRa) systems
In order to enhance the transcription activation power of dCas9, few CRISPR activation systems have been developed and are expressed in viral vectors such as adeno-associated virus (AAV) vectors or lentiviral vectors.
dCas9-VP64-p65-Rta
The first CRISPR activation system is the dCas9-VP64-p65-Rta system, abbreviated as dCas9-VPR. This system entails attaching the VP64 transcriptional activator to the C-terminus of dCas9, followed by the addition of other transcription factors, p65, and Rta, to the C-terminus of dCas9-VP64, as shown in below Fig (Lin et al., 2015; Riedmayr et al., 2022).
For the expression of dCas9-VPR system, an all-in-one vector is used, which contains both gRNA cloning sites under the U6 promoter and a CMV promoter-driven dCas9-VP64-p65-Rta expression cassette. If multiple genes are required to be targeted, then multiple gRNAs are cloned in the vector.
Once expressed, all three transcription factors — VP64, p65, and Rta are targeted to the same gene specified by the gRNA sequence (Moreno et al., 2018; McDougald et al., 2019). Using all three transcription factors, rather than only Vp64 or p65, leads to a higher expression level of the targeted genes. Studies have shown that when dCas9 was used to target various genes, they exhibited significantly higher levels of expression with dCas9-VPR than with dCas9-VP64 alone. To enhance the expression of multiple genes within a single cell utilizing the dCas9-VPR system, multiple sgRNAs are introduced into the cell.
The dCas9-VPR system has been used to activate the genes: neurogenin 2 and neurogenic differentiation 1, resulting in the differentiation of induced pluripotent stem cells into neurons that can be used for developing treatments for various neurodegenerative conditions, including Alzheimer’s disease, stroke, and Parkinson’s disease. The system has also been used to activate the Myod1 gene in fibroblasts to successfully reprogram them into myocytes (Dominguez et al., 2016; Nihongaki, 2017; Chen and Qi, 2017; Omachi and Miner, 2021).
Synergistic Activation Mediator (SAM)
The second way to enhance the transcription activation power of dCas9 is by developing the synergistic activation mediator, abbreviated as SAM. This system is built upon the basic dCas9-VP64 structure but includes a modified sgRNA that incorporates two 138-nucleotide (ntd) RNA hairpin aptamers (Xiong et al., 2016; Vad-Nielsen et al., 2018; Hunt et al., 2021). The RNA hairpin aptamers form the binding sites for dimers of the bacteriophage MS2 coat proteins. MS2 coat proteins can further recruit additional activators, such as p65 and the activation domain of human heat shock factor 1 (HSF1).
For the expression of the SAM system, separate CRISPR vectors are required. One vector contains desired gRNA cloning sites under the U6 promoter and a CMV promoter-driven dCas9-VP64 expression cassette. The desired gRNA sequences are modified to include two 138-ntd hairpin RNA aptamers, which form binding sites for the bacteriophage MS2 coat proteins. If multiple genes are required to be targeted, then multiple modified gRNAs are cloned in the vector.
And the second vector drives the expression of MS2, p65, and HSF-1 proteins under CMV promoters.
When cells are co-transduced with these vectors, the dCas9 enzyme fused with VP64 forms a complex with the desired gRNA and binds to the upstream regions proximal to a promoter of the target gene. The RNA hairpin aptamers of gRNA recruit MS2 proteins, which further recruit p65 and HSF-1 proteins. These transcriptional activators then recruit other transcription factors and RNA polymerase to the promoter to initiate transcription of the gene.
Through synergistic interactions among the VP64, p65, and HSF1 activation domains, the dCas9-SAM system has the potential to amplify gene expression from 10 to multiple thousand-fold. The dCas9-SAM technique is being used by scientists to eradicate HIV-1 in infected organisms by reactivating the latent HIV gene by over-expressing viral proteins in the HIV host cells. Over-expression of viral proteins substantially triggered apoptosis of HIV-infected T cells for their destruction (Saayman et al., 2015; Zhang et al., 2015). Therefore, CRISPRa can provide an alternative therapeutic approach to excise HIV DNA from host DNA in HIV-infected T cells (discussed in Part 44).
Supernova Tagging System
The third way of enhancing the transcription activation of dCas9 is by using the Supernova Tagging system. In this system, the dCas9 protein is modified to be linked with the SunTag. The SunTag is a repeating polypeptide array with multiple copies of GCN4 peptide, which can recruit multiple copies of antibodies that are attached to transcriptional factors like VP64 or p65 (Tanenbaum et al., 2014; Konermann et al., 2015). By attaching transcriptional factors to the antibodies, the SunTag-dCas9 activating complex has the potential to amplify the gene expression by more than 50 fold.
Generally, the antibodies used are GCN4 antibodies which can bind to transcriptional factor VP64. For the expression of the Supernova Tagging system, three CRISPR vectors are required.
· One vector drives the expression of dCas9 and SunTag, i.e., multiple copies of GCN4 peptide.
· The second vector is responsible for inducing the expression of both GCN4 antibodies and the transcriptional activator VP64. Also, a Nuclear Localization Signal (NLS) tag is attached to transport the antibodies to the cell nuclei.
· And the third vector drives the expression of sgRNA.
When the cells are cotransduced with these vectors, the dCas9 enzyme fused with GCN4 peptides forms a complex with the desired gRNA and binds to the upstream regions proximal to a promoter of the target gene. Subsequently, the expressed GCN4 antibodies fused to the transcriptional factors VP64 bind to the GCN4 peptide fused to the dCas9. The attached multiple transcriptional activators then activate the target gene expression.
Studies have shown that the antibodies successfully bound to SunTag polypeptides, and transcriptional activator VP64 activated target CXCR4 gene expression in K562 cell lines by 50-fold. Also, the dCas9-SunTag-VP64 system has been used to reactivate latent HIV-1 transcription in infected human T-cell lines (Ji et al., 2016; Kazi and Biswas, 2021).
A study has shown that when used with dCas9, the VPR, SAM, and SunTag activators are most effective at boosting gene expression across a range of fruit fly, mouse, and human cell lines.
Gene Therapy using CRISPRa
Gene therapy using the CRISPR activation system is under clinical trials. Healthy mice and humans have two copies of genes: one inherited from each parent. A defect in one copy can abolish gene function, leading to a reduced amount of protein and, consequently, human disease, a condition termed as haploinsufficiency. More than 660 genes are currently estimated to cause diseases due to haploinsufficiency, thus, leading to a wide range of phenotypes that include metabolic disorders, immunological diseases, neurological diseases, developmental disorders, kidney disease, cancer, infertility, and many others.
Let’s take the example of obesity which is caused by haploinsufficiency of either the transcription factor Sim1 or the melanocortin 4 receptor (Mc4r) gene. Using a sgRNA, when dCas9 fused to an activator is targeted to either the Sim1 promoter or its ~270 kb distant enhancer in the neuronal cells, it up-regulated Sim1 expression and rescued Sim1-mediated obesity in haploinsufficient mice (Matharu et al., 2019; Montefiori and Nobrega, 2019). On the other hand, targeting Mc4r promoter by means of a similar CRISPRa approach reduces weight gain in obese mice.
The other example of CRISPRa is eliciting antitumor immunity. The tumor antigens might not be expressed at levels sufficient to elicit an effective T cell-mediated response against cancer cells. In other cases, cancer cells downregulate antigen presentation to escape immune recognition. But delivering the CRISPRa system into tumor cells via AAV or lentiviral vectors activates the expression of endogenous genes, thereby enhancing the tumor antigens’ presentation. This leads to increased antitumor immune responses like enhanced T cell recruitment to kill the tumor cells (Wang et al., 2019).
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