Three-Component Repurposed Technology for Enhanced Expression: Highly Accumulable Transcriptional Activators via Branched Tag Arrays

Abstract

In the past few years, several types of artificial transcriptional activator, based on CRISPR-Cas9, have been developed and refined. Of these, in synergistic activation mediator and SunTag systems, the effector proteins, expressed in trans , can be recruited to the target sites via the MS2 RNA-binding system and GCN4-scFv antibody system, respectively. Here, we report a strong transcriptional activation system achieved by fusing GCN4 repeat to MS2 coat protein to accumulate numbers of activators, fused to scFv antibodies. By targeting the CDH1 gene, we show that our novel system, named “TREE,” results in a greater effect of activating exogenous reporter and endogenous gene. Moreover, by targeting another gene, RANKL , we consistently show the superiority of the TREE system with fewer single-guide RNAs compared to conventional systems. Our TREE system is a promising tool for transcriptional activation and can potentially contribute to other dCas9-mediated technologies such as epigenome editing and chromosome visualization.

Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants

“…While allowing efficient changes in gene expression in various cellular contexts including human, mouse, and Drosophila models, the performance of each of the above mentioned systems varies greatly depending on target loci and cell types [ 133 ]. Many more variants have been designed since, including the three-component repurposed technology for enhanced expression (TREE) system, which combines SunTag with the RNA tethering system used by SAM in a tree-resembling structure [ 134 ].…”

Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

“…For instance, the CRISPR-Sirius system can recruit 16 effector proteins through an engineered sgRNA scaffold with eight MS2 aptamers ( Figure 1 B) [ 47 ]. The TREE system, which combines the MS2 and SunTag systems, has the ability to recruit even more effector proteins ( Figure 1 B) [ 48 ]. With more deaminases recruited to genomic regions of interest, the editing window could be enlarged.…”

Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems

“…Three-component repurposed technology for enhanced expression (TREE) is a strong transcriptional activation system, which combines attraction of effectors by RNA aptamers and by protein tags ( Kunii et al, 2018 ). It consists of dCas9-VP64, sgRNA with two MS2 aptamers, SunTag fused with MS2 protein, and scFv-effector.…”