dCas9 regulator to neutralize competition in CRISPRi circuits

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 sgRNAs9 demand, mitigating competition in CRISPRi-based logic gates. The regulator9s 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 gate9s 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.