Kunii Atsushi, Hara Yoshihiro, Takenaga Mitsumasa, Hattori Naoko, Fukazawa Takuya, Ushijima Toshikazu, Yamamoto Takashi, Sakuma Tetsushi
1 Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University , Hiroshima, Japan .
2 Division of Epigenomics, National Cancer Center Research Institute , Tokyo, Japan .
CRISPR J. 2018 Oct;1(5):337-347. doi: 10.1089/crispr.2018.0009.
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 , 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 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, , 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.
在过去几年中，基于CRISPR-Cas9的几种人工转录激活剂已得到开发和优化。其中，在协同激活介质和SunTag系统中，分别通过MS2 RNA结合系统和GCN4-scFv抗体系统，可将在 中表达的效应蛋白招募至靶位点。在此，我们报告了一种强大的转录激活系统，该系统通过将GCN4重复序列与MS2外壳蛋白融合以积累与scFv抗体融合的激活剂数量来实现。通过靶向 基因，我们表明我们名为“TREE”的新系统对外源报告基因和内源基因具有更强的激活作用。此外，通过靶向另一个基因 ，我们始终证明与传统系统相比，TREE系统在使用更少的单向导RNA时具有优越性。我们的TREE系统是一种有前途的转录激活工具，可能有助于其他dCas9介导的技术，如表观基因组编辑和染色体可视化。
