Nielsen Alec A K, Voigt Christopher A
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
Mol Syst Biol. 2014 Nov 24;10(11):763. doi: 10.15252/msb.20145735.
Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on-target repression (56- to 440-fold) and negligible off-target interactions (< 1.3-fold). These gates were connected to build larger circuits, including the Boolean-complete NOR gate and a 3-gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance).
基因回路需要许多调控元件才能在细胞中实现信号处理或执行算法。一种具有潜在扩展性的方法是使用dCas9，它通过RNA:DNA碱基配对的可编程性，利用小向导RNA（sgRNA）来抑制基因位点。为此，我们使用dCas9并设计sgRNA来构建转录逻辑门，并将它们连接起来以在活细胞中进行计算。我们通过设计五个被相应sgRNA抑制的合成大肠杆菌σ70启动子构建了一组非门，并且这些相互作用之间没有相互串扰。这些sgRNA表现出高靶向抑制（56至440倍）且脱靶相互作用可忽略不计（<1.3倍）。这些门被连接起来构建更大的回路，包括布尔完备或非门和一个由四层sgRNA组成的三闸门回路。通过设计输出sgRNA靶向大肠杆菌转录因子（malT），将合成回路连接到天然大肠杆菌调控网络。这将合成回路的输出转化为细胞表型（糖利用、趋化性、噬菌体抗性）的开关。
