Overcoming Leak Sensitivity in CRISPRi Circuits Using Antisense RNA Sequestration and Regulatory Feedback

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.