Resource competition in CRISPRa genetic circuits

CRISPR-mediated gene activation (CRISPRa) allows concurrent regulation of transcription of many gene targets and has found widespread applications in genome-wide screening, bioproduction, and therapeutics. In principle, multiple gene targets can be regulated independently by choosing orthogonal scaffold RNA (scRNA); in practice, this is not the case due to competition for dCas9 and activator protein. While prior studies have extensively characterized competition effects in CRISPRi circuits such an investigation in CRISPRa circuits is still lacking. In this paper, we investigate the on-target and off-target effects of competition for the two resources during CRISPRa. We observe a biphasic response in the on-target response of a CRISPRa module, wherein increased expression of scRNA leads to target repression instead of activation. The addition of a competitor further leads to the repression of any other CRISPRa system, showing the lack of modularity of concurrent CRISPRa systems. These effects are significant even when both resources are expressed at their maximum tolerable limit. We finally show that competition can also be used to design the response of a CRISPRa module by reducing leakiness while keeping the same maximal activation. The results from this study can significantly affect the interpretation of data obtained when using multiple CRISPRi/a modules and can be used as a predictive tool to determine how the operation of any such module in a concurrent configuration is different from the one expected in isolation.