CRISPRgates: 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 128 synthetic ‘barcoded’ promoters in E. coli, 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. We take a multiplexed approach using ‘randomized’ promoter barcodes to demonstrate orthogonality of the ‘CRISPRgates’ on a large scale. This method is immediately transferrable to many different CRISPR nucleases, including Cas9, Cas12a, Cas13a, and CasX, and we use it to study and contrast the behavior of these different systems and probe their properties, including the crRNA structure and PAM site sequence. We then explore how these orthogonal circuit elements can be exploited to create complex genetic circuits. These gates function as transcriptional NOT gates but are Boolean-complete and can in principle be assembled to produce time-dynamic gene circuits.