The controlled binding of the catalytically dead CRISPR nuclease (dCas) to DNA can be used to create complex, programmable transcriptional genetic circuits, a fundamental goal of synthetic biology. This approach, called CRISPR interference (CRISPRi), is advantageous over existing methods because the programmable nature of CRISPR proteins in principle enables the simultaneous regulation of many different targets without crosstalk. However, the performance of dCas-based genetic circuits is limited by both the sensitivity to leaky repression within CRISPRi logic gates and retroactive effects due to a shared pool of dCas proteins. By utilizing antisense RNAs (asRNAs) to sequester gRNA transcripts as well as CRISPRi feedback to self-regulate asRNA production, we demonstrate a mechanism that suppresses unwanted repression by CRISPRi and improves logical gene circuit function in Escherichia coli. This improvement is particularly pronounced during stationary expression when CRISPRi circuits do not achieve the expected regulatory dynamics. Furthermore, the use of dual CRISPRi/asRNA inverters restores the logical performance of layered circuits such as a double inverter. By studying circuit induction at the single-cell level in microfluidic channels, we provide insight into the dynamics of antisense sequestration of gRNA and regulatory feedback on dCas-based repression and derepression. These results demonstrate how CRISPRi inverters can be improved for use in more complex genetic circuitry without sacrificing the programmability and orthogonality of dCas proteins.
Keywords: CRISPRi; antisense; circuit; inverter; sequestration; single-cell.