Digital logic circuits in yeast with CRISPR-dCas9 NOR gates.
Digital logic circuits in yeast with CRISPR-dCas9 NOR gates.
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DOI：
10.1038/ncomms15459
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发表时间：
2017-05-25
影响因子：
16.6
通讯作者：
Klavins E
中科院分区：
文献类型：
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作者：
Gander MW;Vrana JD;Voje WE;Carothers JM;Klavins E
自然的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，因为常用的组件会转录泄漏，不会任意互连或没有数字响应。在这里，我们在酿酒酵母中设计了基于dCas 9-Mxi 1的NOR门，允许任意连接和大型遗传电路。因为我们使用了染色质重塑剂Mxi 1，所以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门的组合库，可以直接将向导RNA（gRNA）输入转换为gRNA输出，使这些门能够“连接”在一起。我们用多达七个gRNA构建了逻辑电路，包括多达七层的抑制级联。建模预测NOR门具有有效的零转录泄漏，解释了电路中有限的信号退化。我们的方法实现了迄今为止最大的真核基因电路，并将成为大型合成细胞决策系统的基础。常用的遗传成分的泄漏可能会使复杂合成电路的构建变得困难。在这里，作者构建了NOR门架构，使用dCas 9融合到染色质重塑器Mxi 1，可以连接在一起形成复杂的电路。
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. The leakiness of commonly used genetic components can make the construction of complex synthetic circuits difficult. Here the authors construct NOR gate architecture, using dCas9 fused to the chromatin remodeller Mxi1, that can be wired together into complex circuits.