CRISPRi-mediated gene repression allows simultaneous control of many genes. However, despite highly specific sgRNA-promoter binding, multiple sgRNAs still interfere with one another by competing for dCas9. We created a dCas9 regulator that adjusts dCas9 concentration based on sgRNAs' demand, mitigating competition in CRISPRi-based logic gates. The regulator's performance is demonstrated on both single-stage and layered CRISPRi logic gates and in two common E. coli strains. When a competitor sgRNA causes between two and ~25 fold-change in a logic gate's input/output response without dCas9 regulator, the response is essentially unchanged when the regulator is used. The dCas9 regulator thus enables concurrent and independent operation of multiple sgRNAs, thereby supporting independent control of multiple genes.

中文翻译：

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dCas9调节剂可消除CRISPRi电路中的竞争

CRISPRi介导的基因阻抑允许同时控制许多基因。但是，尽管高度特异性地结合了sgRNA-启动子，但多个sgRNA仍然通过竞争dCas9相互干扰。我们创建了一个dCas9调节剂，可根据sgRNA的需求调节dCas9的浓度，从而减轻基于CRISPRi的逻辑门的竞争。在单级和分层CRISPRi逻辑门以及两种常见的大肠杆菌菌株中均展示了调节器的性能。当竞争对手的sgRNA在没有dCas9调节器的情况下导致逻辑门的输入/输出响应变化介于2到25倍之间时，使用调节器时，响应基本上没有变化。因此，dCas9调节剂使多个sgRNA并发且独立运行，从而支持多个基因的独立控制。