Improved CRISPRi gene circuit function via context-sensitive antisense RNA sequestration

By using the binding of the catalytically-dead CRISPR protein dCas12a, we can create programmable gene circuit elements in E. coli. These ‘CRISPRgates’ are simple NOT gate elements which can target genes or other CRISPRgate elements and in principle can be combined to create complex genetic circuits, a fundamental goal of synthetic biology. While natural transcription factors have built-in advantages for transcriptional regulation (e.g. cooperativity), they are not programmable or individually orthogonal and a limited number of them exist. Repression with CRISPR is advantageous because we can in principle repress many different targets simultaneously without crosstalk. However, such gene circuit elements behave poorly when placed in series due to signal loss that occurs due to leaky repression (e.g. NOT NOT NOT != NOT) and retroactivity effects due to a shared pool of Cas proteins. By utilizing antisense RNAs to sequester guide RNA transcripts in combination with appropriate spatial arrangement of gene circuit nodes, we demonstrate a mechanism to suppress leaky CRISPRi repression and restore logical gene circuit function when elements are used in series.