3/17/2023 0 Comments Vp64 and srdxTherefore, it is crucial to develop CRISPRa technologies that take into account both the efficiency of gene activation and side effects for in vivo applications. On the other hand, ubiquitous expression of Cas9 18 and dCas9-VP64 6 in mice is not overtly toxic, suggesting that neither dCas9 nor VP64 is toxic in vivo, and that high expression of either p65 or Rta, or both, may be responsible for the observed toxicity. Also, in mice, ubiquitous expression of VPR during development and expression in inhibitory neurons are toxic 17. For example, it has been reported that VPR and SAM are toxic when highly expressed in Drosophila with a strong promoter 16. Specifically, several studies have reported on the in vivo toxicity of CRISPRa components. Based on a different concept from transcriptional inducers, dCas9 fused to epigenetic modifiers such as p300, histone acetylase 14 and Tet1, a CpG DNA demethylase, 15 have also been used to activate endogenous genes.Īlthough these studies have significantly contributed to the development of CRISPRa technology, most of them have been conducted in in vitro and ex vivo cell culture systems, and there are still challenges regarding in vivo applications. More recently, additional transcriptional activators such as p65, Rta, and HSF1 have been identified, and CRISPRa-VPR 2, SAM 11, SPH 12 and TREE 13 systems have been developed by combining multiple transcriptional activators. In a similar approach, VP64-dCas9-VP64, in which one VP64 was added to the N-terminus of dCas9-VP64, exhibited increased efficiency of transcriptional activation 1. Subsequently, protein tagging systems called Suntag (dCas9-Suntag-VP64) 10 and MS2-MCP (dCas9-VP64 + MCP-VP64) 11 were developed to increase the number of VP64s at the same locus and enhance activation efficiency. The first generation version of CRISPRa, dCas9-VP64, was developed using VP64, a transcriptional activator, and dead Cas9 (dCas9) 7, which has no nuclease activity, allowing activation of guide RNA (gRNA) -targeted endogenous genes 8 9. CRISPRa has been used in broad fields of research, including direct cell reprogramming by controlling master transcription factors that regulate cell lineage, 1– 3 cancer modeling by activating oncogenes 4, and therapeutic approaches by activating disease-modifying genes 5 and genes deficient due to haploinsufficiency 6. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.ĬRISPR/Cas9-mediated activation (CRISPRa)-based regulation of gene expression is a powerful tool for understanding complex biological phenomena, because it allows for the simultaneous regulation of multiple genes. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. We found that MS2-MCP-scaffolded VP64 enhanced dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Although CRISPRa approaches based on VP64 have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena.
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