Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal context. However, embedding a biosensor into a gene coding sequence may unpredictably alter endogenous gene regulation. To address this challenge, we developed an approach to integrate genetic biosensors into endogenous genes without modifying their coding sequence by inserting into their terminator region single-guide RNAs that activate downstream circuits. Sensor dosage responses can be fine-tuned and predicted through a mathematical model. We engineered a cell stress sensor and actuator in CHO-K1 cells that conditionally activates antiapoptotic protein BCL-2 through a downstream circuit, thereby increasing cell survival under stress conditions. Our gene sensor and actuator platform has potential use for a wide range of applications that include biomanufacturing, cell fate control and cell-based therapeutics.

合成生物学旨在通过实施响应细胞状态变化的遗传电路来改变细胞行为。使用成簇的规则间隔的短回文重复序列和 Cas9 将遗传生物传感器整合到内源基因编码序列中，从而能够在适当的染色体环境中询问基因表达动态。然而，将生物传感器嵌入基因编码序列可能会不可预测地改变内源基因调控。为了应对这一挑战，我们开发了一种方法，通过在终止子区域插入激活下游电路的单向导RNA，将遗传生物传感器整合到内源基因中，而无需修改其编码序列。传感器剂量响应可以通过数学模型进行微调和预测。我们在 CHO-K1 细胞中设计了一种细胞应激传感器和执行器，通过下游电路有条件地激活抗凋亡蛋白 BCL-2，从而提高细胞在应激条件下的存活率。我们的基因传感器和执行器平台具有广泛的应用潜力，包括生物制造、细胞命运控制和基于细胞的治疗。