Department of Electrical Engineering, University of Washington, Seattle, Washington 98195, USA.
Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
Nat Commun. 2017 May 25;8:15459. doi: 10.1038/ncomms15459.
Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses. We built a combinatorial library of NOR gates that directly convert guide RNA (gRNA) inputs into gRNA outputs, enabling the gates to be 'wired' together. We constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Modelling predicted the NOR gates have effectively zero transcriptional leak explaining the limited signal degradation in the circuits. Our approach enabled the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, cellular decision-making systems.
自然遗传电路使细胞能够做出复杂的数字决策。然而,在真核生物中构建同样复杂的合成电路仍然很困难,因为常用的元件在转录水平上会泄漏,不能任意连接,或者没有数字响应。在这里,我们在酿酒酵母中设计了基于 dCas9-Mxi1 的 NOR 门,允许任意连接和构建大型遗传电路。由于我们使用了染色质重塑酶 Mxi1,我们的门显示出最小的泄漏和数字响应。我们构建了一个 NOR 门的组合文库,该文库可以将向导 RNA(gRNA)输入直接转换为 gRNA 输出,从而将门“连接”在一起。我们构建了具有多达七个 gRNA 的逻辑电路,包括具有多达七个层的抑制级联。模型预测 NOR 门的转录泄漏率有效为零,这解释了电路中信号的有限衰减。我们的方法实现了迄今为止最大的真核基因电路,并将为大型合成细胞决策系统奠定基础。