Despite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

中文翻译：

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表征，建模和减轻哺乳动物细胞中基因表达的负担

尽管在基因组工程方面有最新进展，但是哺乳动物细胞中遗传电路的设计仍然十分缓慢，并且充满了莫名其妙的失败。在这里，我们证明了对有限转录和翻译资源的竞争会动态地偶联原本相互独立表达的外源基因，从而导致性能下降，并导致预期功能与实际功能之间的差异。我们还显示，当遗传有效载荷在宿主细胞中表达时，内源基因的表达同样受到影响。遵循资源感知的数学模型以及我们的实验发现，即转录后监管者具有很大的资源再分配能力，我们确定和工程设计基于天然和合成的基于miRNA的非相干前馈环（iFFL）电路，以减轻基因表达负担。这些电路的实现采用内源性miRNA作为工程化iFFL装置不可或缺的组成部分的新颖用途，这种通用的混合设计允许以最小的资源需求跨不同的细胞系实现减负。这项研究为上下文感知的预测和改进提供了基础。体内合成电路的性能，为哺乳动物细胞中更合理的合成构建体设计铺平了道路。