In this method, dCas9 is fused to CBP, an histone acetyltransferase domain capable of rearranging chromatin structure. These newer methods have not yet been rigorously compared to other CRISPR activators, but their initial results show promise for CRISPR-based gene activation. While SAM, SunTag, and VPR have become the most popular methods for CRISPR activation, research in the area has continued to develop. This streamlines its delivery, making it a common choice for CRISPR activation. An advantage to this method compared to other notable CRISPR activators is that it requires a fusion protein, rather than relying on a two-component system dependent on gRNA design (SAM) or peptide design (SunTag). Its activation levels are similar to that of SunTag. Generally, VPR is found to have significantly higher activation levels than the initial dCas9-VP64 activator, but lower levels compared to the SAM system. These transcriptional activators work in tandem to recruit transcription factors. Srdx transcription Activator#This complex consists of the VP64 activator used in other CRISPR activation methods, as well as two other potent transcriptional activators (p65 and Rta). VPR fuses a tripartite complex with dCas9 to activate transcription. One drawback of this method is its construction: it relies on antibody chains, which are relatively large and are not expressed consistently throughout cells. SunTag performs better than first generation activators while showing lower activation levels than SAM. By having multiple copies of VP64 at each loci of interest, this allows more transcriptional machinery to be recruited per targeted gene. Rather than using a single copy of VP64 per each dCas9, SunTag uses a repeating peptide array to fused with multiple copies of VP64. In cases of multiplex gene regulation (activating multiple genes at once), however, SAM exhibits activation levels comparable to other popular activation methods (VPR and SunTag) ( Chavez et al., 2016). When targeting single genes, SAM consistently shows the highest levels of gene activation compared to other CRISPR activators, making it a popular method for gene activation experiments. These MS2 proteins then recruit additional activation domains (HS1 and p65). This is done through creating a dCas9/VP64 fusion protein engineered with aptamers that bind to MS2 proteins. SAM uses specially engineered sgRNAs to increase transcription. Although other methods have been able to achieve much higher activation, dCas9-VP64 is great for experiments that requires modest gene activation. While it requires a relatively simple construct, it exhibits modest levels of gene activation, with some genes experiencing around 2-fold activation levels. This can be used to activate transcription during either initiation or elongation, depending on which sequence is targeted.ĭCas9-VP64 activation is generally thought of as the “first generation” CRISPR activator. Guided by dCas9, VP64 recruits transcriptional machinery to specific sequences, causing targeted gene regulation. dCas9-VP64ĬRISPR activation can occur through fusing dCas9 with VP64, a strong transcriptional activation domain. SunTag, SAM, and VPR have all shown significant improvements upon the initial dCas9-VP64 method, so there are multiple options to choose from when looking to activate genes across diverse cell lines. CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others re-engineer components of Cas systems themselves. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. To target specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest. Most popular methods for CRISPR activationĬRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease ability, to bind to genes without editing the genome (Qi et al., 2013). In the years that followed, innovative methods greatly improved CRISPRa, expanding its practicality and popularity in research ( Tanenbaum et al., 2014, Konermann et al., 2015, Chavez et al., 2015). Gene activation by dCas9, also referred to as CRISPRa, was initially published in 2013 ( Bikard et al., 2013, Perez-Pinera et al., 2013). The development of CRISPR/Cas systems, however, greatly improved the simplicity of gene activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only require changing the programmable guide RNA. When using zinc finger proteins or TALE proteins, proteins had to be re-engineered for each gene, making wide-scale gene activation seem next to impossible. Prior to the discovery of CRISPR/Cas systems, gene activation across multiple loci was an arduous process.
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