Programmable and Orthogonal Gene Circuit Elements

While CRISPR may be better-known as a gene editing tool, it can also be used as a tool for selective repression of genes using a catalytically-dead CRISPR nuclease. Here, we exploit both the freedom in the CRISPR identification region as well as the freedom in promoter design to create a set of 64 synthetic ‘barcoded’ promoters, each of which is keyed to a specific CRISPR guide RNA. These synthetic promoters can then be used to drive chosen genes in modular fashion. By driving GFP, we measure the on/off ratio of these so-called CRISPRgate circuit elements. We then take a multiplexed approach using ‘randomized’ promoter barcodes in order to demonstrate orthogonality of the CRISPRgates on a large scale. This is done using a sacB/tetA cassette, a counterselection scheme which slows cell growth in the presence of sucrose and fusaric acid. Cells with matched CRISPR RNA and promoter barcodes preferentially survive, and by using multiplexed sequencing, we demonstrate that these CRISPRgate circuit elements are indeed orthogonal on a massive scale. We then explore how these orthogonal circuit elements can be exploited to create complex genetic circuits.