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26
2024
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08
South University of Science and Technology Chen Wei/Cui Huanhuan Cooperate in the Latest Nature Sub-journal
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CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disturbed regions (IDRs). However, the underlying mechanism remains controversial.
On August 22, 2024,Southern University of Science and TechnologyChen Wei andCui HuanhuanCommon CommunicationInNature Communications Published online entitled“Specific multivalent molecules boost CRISPR-mediated transcriptional activation”The research paper,The studyA general platform for efficient gene activation was developed and provides important insights into the multivalent molecular enhancer transcriptional activator based on CRIPSR.
The study was conducted by combining 12 knownIDRsFusing into dCas9-VP64 activators was studied, of which only seven could enhance activation, although they were independent of phase separation ability. In addition, the modular structure domain (MDs) is another class of multivalent molecules that, although by themselves cannot enhance the activity of dCas9-VP64, when compared with dCas9-VP64-IDRWhen bound, transcriptional activation can be significantly enhanced. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, it was found that the optimal, rather than the maximum, cis-trans synergistic performance achieved the most stable activation.Finally, targeted promoter-enhancer pairs produce synergistic effects that can be further amplified by enhancing chromatin interactions.
Gene transcription is regulated by dynamic interactions between DNA-binding transcription factors (TFs) and cis-regulatory elements.TFsIt consists of DNA-binding domains (DBDs), which have a well-defined protein structure that recognizes specific DNA motifs, and activation domains (ADs), which recruit co-activators and usually contain intrinsically disordered regions (IDRs) that lack regular functional structure.TFsIdentifiedcis-regulatory elementincluding the proximal promoter and distal enhancer where transcription initiation occurs,Distal enhancers via higher orderTFsCoactivator complex-mediated engagement of the target promoter with a remote chromatin loop enhances transcription.
In order to manipulate gene expression and reconnect endogenous transcriptional networks, synthetic zinc finger-based (ZFs) and transcriptional activator-like effectors (TALEs) have been designedTFs.Recently, a similar system based on nuclease killed Cas9 (dCas9) was developed, called CRISPRa. The first generation CRISPRa system utilizes VP64 AD fused to dCas9 to activate transcription of target genes by a guide RNA (gRNA) complementary to a promoter sequence. In order to achieve higher efficiency, subsequent improvements include modification of the fusion AD or gRNA design. For example, the VPR system fuses the VP64, p65, and Rta activation domains onto dCas9 for synergistic activation. The SunTag system contains up to 24 GCN4 peptide repeat docking sites on dCas9, thereby recruiting multiple activator copies to a specific genomic site by the fused GCN4 antibody. CRISPR/gRNAdirected synergistic activation mediator (SAM) systems utilizing modified gRNARecruitment of multiple activation domains by RNA ligands. The purpose of all these modifications is to increase the local concentration of transcriptional activators at the genomic site of interest.However, these approaches often require multiple bulky fusion partners, potentially hindering delivery, particularly in therapeutic applications.
Liquid-liquid phase separation (LLPS) is a common physical phenomenon, proteinIDROr modular domains (MDs) can drive the formation of biomolecular aggregates or membrane-free organelles, thereby partitioning biochemical reactions.in the process of gene transcription,TFsand its coactivators (usually includingIDR) regulates the formation of transcriptional aggregates by homotypic or heterotypic multivalent interactions. Transcriptional aggregates on superenhancers have been shown to drive transcription of highly active genes. Furthermore, the IDR of FUS protein was associatedTetR-VP16The fusion results in increased phase separation and transcriptional activation. Recently, the IDRs of NUP98 and FUS have also been reported to enhance the transcriptional activation potential of dCas9-VPR. However, two recent studies have shown that optimized multivalent interactions, rather than LLPS itself, enhance transcriptional activation.Although the complex role of LLPS in transcriptional regulation remains controversial, the use of multivalent interactions offers hope for the further development of enhanced transcriptional activation systems.
In order to use multivalent interactions to enhance the basis.CRISPRof transcriptional activation, the study screened 12IDR.Although all theseIDRBoth are known for their ability to induce phase separation, but only 7 can enhance activation. toDCas9-VP64-FUSAs an example, the study demonstrated that cells, with or without a dCas9 microscopic focus formed by phase separation, showed efficient transcriptional activation of target genes. In addition to the IDR, several MDs have been studied, another class of molecules that promote multivalent interactions but have not been associated with transcriptional regulation. Interestingly, while MDs by themselves did not enhance dCas9-VP64 activity, they were associated with dCas9-VP64-IDRThe fusion will further enhance transcriptional activation. In addition, by changing the number of gRNA binding sites, and dCas9-VP64 with differentIDRAnd MDs fusion, the study found that the best cis-anti synergy (rather than the maximum cis-anti synergy) is essential for strong activation. Finally, by simultaneously targeting the promoter-enhancer pair, the studied enhanced activation system shows a synergistic effect that relies heavily onIDRand further amplification by enhancing chromatin interactions.Therefore, research has developed a versatile platform not only for efficient gene activation, but also for the study of underlying molecular mechanisms.
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