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

ABSTRACT: 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 sup

SUBMITTER: Specht DA 

PROVIDER: S-EPMC9486968 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature