Background

By controlling the gene expression in living cells, the function of cells can be reprogramed for a variety of purposes. For example, if a plant cell is invaded by a pathogen, it would be advantageous for the cell to express genes related to antimicrobial metabolite production, and this expression should only occur in the presence of pathogenic microbes rather than beneficial microbes. A synthetic genetic circuit is one of the man-made molecular tools to control the gene expression of cells. Synthetic circuits use logic gates analogous to those used in electronics to generate outputs. Basic logic gates include YES, NOT, AND, OR, NOR, etc. and these can be used to control when and where a selected set of genes is active. For instance, in an AND gate, an output reporter gene will only be expressed if both input modules A and B are present at the same time (see ). If applied to the previous example, a potential circuit could be that the genes encoding antimicrobial proteins would be expressed in the cell only if both A and B input modules are triggered by invasion of a specific pathogenic microbe. Over the years, numerous versions of genetic control circuits that recapitulate individual basic logic gates have been reported. However, there are a number of limitations on these circuits, such as poor scalability, low reusability, incompatibility with particular gates.

Technology Overview

Researchers at North Carolina State University have utilized the CRISPR-Cas9 system to create synthetic genetic circuits. Their method uses crRNA-tracrRNA-based binary logic gates and a nuclease-dead version of Cas9 fused to a transcriptional activation or repression domain, dCas9-AD or dCas9-RD, acting as a sequence-specific, crRNA-guided, synthetic transcription factor that activates or represses target gene expression in a highly regulated manner. By using different promoters to drive the expression of the crRNA and tracrRNA in a crRNA-tracrRNA pair, and by combining several of those orthogonal pairs, molecular circuits capable of complex logic operations can be easily designed to control the expression of one or more target genes.

To overcome the scalability problem often seen in combinatorial larger circuits, the researchers developed multiple modified, non-crossreacting crRNA:tracrRNA pairs that work independently and in parallel with other crRNA:tracrRNA pairs in an orthogonal manner through a single type of Cas9 in the context of multi-gate synthetic genetic devices. For example, the researchers have built a synthetic genetic circuit that recapitulates a logic AND gate where each component of a crRNA:tracrRNA pair is expressed under a synthetic or native promoter of interest to jointly drive activation or repression of specific genes by targeting dCas9-AD to their promoters only in the tissues, developmental stages, or conditions where/when both crRNA and tracrRNA are co-expressed.

Benefits

Solves promoter “leakiness” issue
Allows for tunable gene expression
Can be used to generate highly programmable traits
Scalable
Straightforward to implement

Applications

The method can potentially be used to tightly regulate expression of one or more gene of interest in any setting (e.g., to induce production of an antimicrobial metabolite upon pathogen infection, to express a toxin specifically in a cancer cell, to trigger stomatal closure or fruit ripening in plants in response to an agri-chemical spray, to regulate plant root architecture, etc.).

Opportunity

NC State University is seeking an industry partner to license and commercialize the technology.

Synthetic Biology Tool for Gene Expression Regulation

Patents

IP Status

Seeking