2 Zinc-finger proteins (ZFPs), transcription activator-like effectors (TALEs) and dCas9 fused with a transcription activation domain (TAD) are three types of ATFs available to mediate not only site-specific DNA recognition but also gene expression regulation. In the eukaryotic cells, 1 the regulation of the expression of the key gene involved in an haploinsufficiency disease could be modulated by artificial transcription factors (ATFs). Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. The results show that the AAV9_3XFLAG-TALE frat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. This AAV9_3XFLAG-TALE frat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. This TALE frat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. We produced an adeno-associated virus 9 (AAV9) coding for a TALE frat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin ( FXN) gene. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. They thus provide a good tool for targeted gene regulation as a therapy. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. The protocol presented here provides a detailed procedure for activating AtPAP1 and repressing AtCSTF64 in Arabidopsis thaliana. When multiple chimeric dCas9-effector fusions are guided to gene regulatory regions via CRISPR gRNAs, they can modulate expression of transcript levels in planta.
#VP64 AND SRDX ACTIVATOR#
First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX). These tools are especially useful for studying gene function and interaction within various regulatory networks. N2 - Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). JF - Methods in molecular biology (Clifton, N.J.)
T1 - Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.
Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs).
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