Kar Shaunak, Bordiya Yogendra, Rodriguez Nestor, Kim Junghyun, Gardner Elizabeth C, Gollihar Jimmy D, Sung Sibum, Ellington Andrew D
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA.
Plant Methods. 2022 Mar 29;18(1):42. doi: 10.1186/s13007-022-00867-1.
The construction and application of synthetic genetic circuits is frequently improved if gene expression can be orthogonally controlled, relative to the host. In plants, orthogonality can be achieved via the use of CRISPR-based transcription factors that are programmed to act on natural or synthetic promoters. The construction of complex gene circuits can require multiple, orthogonal regulatory interactions, and this in turn requires that the full programmability of CRISPR elements be adapted to non-natural and non-standard promoters that have few constraints on their design. Therefore, we have developed synthetic promoter elements in which regions upstream of the minimal 35S CaMV promoter are designed from scratch to interact via programmed gRNAs with dCas9 fusions that allow activation of gene expression.
A panel of three, mutually orthogonal promoters that can be acted on by artificial gRNAs bound by CRISPR regulators were designed. Guide RNA expression targeting these promoters was in turn controlled by either Pol III (U6) or ethylene-inducible Pol II promoters, implementing for the first time a fully artificial Orthogonal Control System (OCS). Following demonstration of the complete orthogonality of the designs, the OCS was tied to cellular metabolism by putting gRNA expression under the control of an endogenous plant signaling molecule, ethylene. The ability to form complex circuitry was demonstrated via the ethylene-driven, ratiometric expression of fluorescent proteins in single plants.
The design of synthetic promoters is highly generalizable to large tracts of sequence space, allowing Orthogonal Control Systems of increasing complexity to potentially be generated at will. The ability to tie in several different basal features of plant molecular biology (Pol II and Pol III promoters, ethylene regulation) to the OCS demonstrates multiple opportunities for engineering at the system level. Moreover, given the fungibility of the core 35S CaMV promoter elements, the derived synthetic promoters can potentially be utilized across a variety of plant species.
如果基因表达能够相对于宿主进行正交控制，那么合成基因回路的构建和应用通常会得到改进。在植物中，可以通过使用基于CRISPR的转录因子来实现正交性，这些转录因子被设计用于作用于天然或合成启动子。复杂基因回路的构建可能需要多个正交调控相互作用，这反过来又要求CRISPR元件的完全可编程性适用于对其设计限制较少的非天然和非标准启动子。因此，我们开发了合成启动子元件，其中最小35S CaMV启动子上游的区域是从头设计的，以便通过编程的gRNA与允许激活基因表达的dCas9融合蛋白相互作用。
设计了一组三个相互正交的启动子，它们可以被与CRISPR调节因子结合的人工gRNA作用。靶向这些启动子的引导RNA表达反过来由Pol III（U6）或乙烯诱导型Pol II启动子控制，首次实现了完全人工的正交控制系统（OCS）。在证明设计的完全正交性之后，通过将gRNA表达置于内源性植物信号分子乙烯的控制之下，将OCS与细胞代谢联系起来。通过乙烯驱动的单株植物中荧光蛋白的比例表达，证明了形成复杂电路的能力。
合成启动子的设计高度可推广到大片段的序列空间，从而有可能随意生成越来越复杂的正交控制系统。将植物分子生物学的几个不同基本特征（Pol II和Pol III启动子、乙烯调节）与OCS联系起来的能力，展示了在系统水平上进行工程设计的多种机会。此外，鉴于核心35S CaMV启动子元件的可互换性，衍生的合成启动子有可能在多种植物物种中使用。
