一種在植物和哺乳動物細胞中高效的由Cas9驅動的基因激活

本文2017年11月20日在線發表於Nature Plants，原文題目為：

A potent Cas9-derived gene activator for plant and mammalian cells

摘要：

cDNA的過表達是最常用的功能獲得的方法，以此來研究基因功能和操縱生物學特徵。然而，由於基因克隆工作量的增加，有限的載體容量，需要多個啟動子和終止子，以及轉基因表達水平的變化，這種方法對於多基因表達具有挑戰性和低效性。合成的轉錄激活因子通過可編程的DNA結合模塊將自發轉錄激活結構域（TAD）連接到內源基因組基因座上的啟動子上，為基因的激活提供了有希望的替代策略。在已知的DNA結合模塊中，通過合成指導RNA和DNA之間的鹼基配對識別特定DNA靶標的dCas9蛋白，性能要優於鋅指蛋白和轉錄激活類效應子，後兩者都通過蛋白質-DNA相互作用來靶向目標。最近，三種高效的基於dCas9的轉錄激活系統，即VPR，SAM和SunTag已經被開發用於動物細胞。然而，在植物細胞中仍然缺乏基於dCas9的轉錄激活系統。在這裡，我們通過植物細胞篩選開發了一種名為dCas9-TV的新型強效dCas9-TAD。與植物和哺乳動物細胞中常規使用的dCas9-VP64激活相比，dCas9-TV賦予單個或多個靶基因更強的轉錄激活。

abstract

Overexpression of complementary DNA represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labour for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at the endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA and DNA, outperforms zinc-finger proteins and transcription activator-like effectors, both of which target through protein–DNA interactions. Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM and SunTag, have been developed for animal cells. However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells. Here, we developed a new potent dCas9–TAD, named dCas9–TV, through plant cell-based screens. dCas9–TV confers far stronger transcriptional activation of single or multiple target genes than the routinely used dCas9–VP64 activator in both plant and mammalian cells.