CRISPR-Cas9 system has been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating double-stranded breaks (DSBs). Original efforts to convert the CRISPR-Cas9 gene editing system into a trans-activator were achieved by fusing a transcriptional activation domain (VP64) to versions of Cas9 that lacked nuclease activity (termed dCas9). This enabled the CRISPR-Cas9 system to transcriptionally activate target genes within the native chromosomal context. Moreover, CRISPR activation offers many advantages over alternative gene overexpression or activation methods. The versatile and efﬁcient in vivo CRISPR-Cas9 mediated gene activation system holds great promise, both as a tool for in vivo biomedical research and as a targeted epigenetic approach for treating a wide range of human diseases.
To convert Cas9 protein from a DNA scissor into a gene activator, it is necessary to disrupt its nuclease activity. Cas9's two nuclease domains, the RuvC and HNH domains, are conserved among several types of nucleases, and each is responsible for cutting one strand of DNA upon binding. Researchers have introduced mutations into these two domains to create a nuclease-deactivated Cas9 (dCas9). This converts the Cas9 into a generic RNA-guided DNA-binding protein. It is then possible to fuse effectors directly to dCas9, which essentially transforms the dCas9-effector fusion into an easily programmable artiﬁcial transcription factor upon being paired with a target-speciﬁc sgRNA. As the two domains are conserved among Cas9s from other bacterial species, this approach provides a general strategy for repurposing orthogonal Cas9s into RNA-guided DNA-binding proteins.
One type of effector that can be fused to dCas9 is a transcriptional activator. There are different forms of these dCas9-activator fusions. For example, researchers fused the ω subunit of RNA polymerase to dCas9 for use in E.coli. This fusion was able to activate reporter gene expression up to 3-fold. In eukaryotic cells, the ﬁrst generation of dCas9 activators consisted of dCas9 fused to the activation domain of p65 or a VP64 activator. The dCas9-VP64 fusion proved more effective than the p65 fusion and has been used more ubiquitously. However, the activation seen in mammalian cells is usually moderate, about 2-fold to 5-fold, on average, using a single sgRNA. This activation can be enhanced by using multiple sgRNAs tiled across the promoter, suggesting that recruiting additional activators to the target gene enhances activation.
Fig 1-1. Various engineered CRISPR activation systems.
The SunTag activation system consists of dCas9 fused to several tandem repeats of a short peptide sequence separated by linkers. The SunTag activator module is an scFv, an engineered portion of an antibody that binds to the peptide repeats in the SunTag array. And the scFv is fused to sfGFP and VP64. Compared to the ~2-fold increase observed with dCas9-VP64 alone, we observed a 50-fold increase at the protein level with dCas9-SunTag for endogenous genes such as the CXCR4 chemokine receptor gene in human erythroleukemia K562 cells. Researchers have also created a tripartite effector fused to dCas9, composed of activators VP64, p65, and Rta (VPR) linked in tandem. These three activators were joined in a deﬁned order to strongly activate genes. Additionally, it can upregulate endogenous gene expression from 5- to 300-fold at the mRNA level compared to a single dCas9-VP64 fusion.
Like the VPR activator, the SAM system employs multiple transcriptional activators to create a synergistic effect. This new sgRNA contains two copies of an RNA hairpin from the MS2 bacteriophage, which interacts with the RNA-binding protein (RBP) MCP (MS2 coat protein). An additional activation module was created by fusing MCP to the p65 transcriptional activator as well as to the activating domain of human heat shock factor 1 (HSF1). MCP binds to MS2 as a dimer, so up to four additional copies of the activation module can be recruited per dCas9-VP64. The SAM system can produce a wide (two- to multiple-thousand-fold) range of enhanced activation of endogenous genes at the mRNA level compared to dCas9-VP64, depending on baseline expression. This includes both protein coding genes and long noncoding RNAs (lncRNAs).
Fig 1-2. Various engineered CRISPR activation systems.
Together, these transcriptional activation systems function across a range of cell types and species and provide many options for transcriptional and epigenetic manipulation. Each strategy comes with its own advantages and disadvantages. For example, while the VPR activator relies on fewer components, it has not yet been validated for larger-scale screens like the SunTag and SAM activators. The high activation levels of the VPR system depended on using a pool of 3 to 4 sgRNAs, making it more difﬁcult to use effectively in genome-wide screens. In addition, there may be cell type-speciﬁc efﬁciency or toxicity issues with each of these technologies. All of these tools are relatively new, and so it will be interesting to compare their efﬁciencies and speciﬁcities directly in a range of cell types and for a variety of genes.
CRISPR activation offers many advantages over alternative gene overexpression or activation methods. One technique to overexpress genes is to clone the open reading frame (ORF) or cDNA of the gene of interest. While, for longer or GC-rich genes, this alone can be technically difﬁcult. In cloning many genes at once using this method, there would be a bias toward smaller and easier-to-amplify genes. Additionally, when cloning from the cDNA, one may be missing physiologically relevant splice variants. Another, alternative approach is to use other engineered transcriptional activators to turn on gene expression. These include zinc ﬁnger (ZFN) effectors and transcriptional activator-like effectors (TALEs) fused to a set of activation domains. As with dCas9 activators, ZFN and TALE activators target speciﬁc sequences of DNA and recruit transcriptional machinery to activate transcription. ZFNs and TALEs rely on protein-DNA interactions.
|Activation method||Activated gene||Engineering||Cost||Throughput of production||Used in pooled genome wide screens||Off-target effects||Limitations in use|
|CRISPR activation||Endogenous||Easy||Low||High||Yes||Minimal||Requries an "NGG" PAM|
|ORF overexpression||Endogenous||Medium to difficult||Low||Low||Yes||None||Longer or GC-rich genes can be difficult to clone|
|TALE or ZFN||Endogenous||Medium to difficult||High||Low||No||Minimal||ZFN requires some engineering to target a sequence|
The main disadvantage of TALEs and ZFN effectors compared to CRISPR-Cas9 is that they require more complicated cloning to assemble, making them less user-friendly overall and not amenable to genome-wide screens. However, the use of TALEs in particular can be advantageous when a particular target area lacks a PAM, since TALEs can be programmed to target any sequence. TALEs are able to distinguish between methylcytosine and cytosine. The methylation status of the targeted site must be taken into account when designing TALEs, which is not the case for Cas9 targeting. Finally, some of the CRISPR activation systems require many components, making them potentially much more complicated to deliver than the one-component ZFN or TALE activators. These characteristics must all be taken into account when deciding which activation system to use.
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