Generation of Layered Transcriptional Circuitry using CRISPR Systems

Applications

Engineered biological circuits that sense input, process logic, and perform output functions have myriad of uses. Among many other things, they can be used for metabolic engineering to produce compounds, engineered to sense inputs such as metabolites or physical conditions of the environment, engineered as targeted therapies against pathological conditions, and used as tools to understand biological pathways and processes.

Problem Addressed

A part of basic transcriptional circuitry is to have expression of a gene product regulate expression of another gene product. A library of these “composable” regulatory devices (having matching input and output types and expression levels) are needed to make complex interconnected functional circuits. A transcriptional framework to make such a library has been missing in the field. Attempts at making libraries using transcriptional effectors fused to DNA binding domains have been limited because of extensive DNA assembly protocols or slow temporal responses of the circuit. CRISPR technology allows for the rapid generation and screening of numerous pairs of circuits (guide RNA and guide RNA controllable promoter) with fast temporal responses, thus, providing a framework to make a library of “composable” regulatory devices that can be layered into complex transcriptional circuitry.

Technology

The core of the technology relies on making promoters regulated by Cas9m (catalytically inactive Cas9) mediated steric blocking of transcription. Flanking either side of RNA Pol II promoters or RNA Pol III promoters with guide RNA targeted sites generates highly specific CRISPR-responsive promoters. Pairs of guide RNA and guide RNA targetable promoter are modular and extensive because they can be generated by simply changing the sequence of the guide RNA and its target sites. These orthogonal regulatory devices can be interconnected to create complex layers of control with multiple inducible input points and any desirable output.

Advantages

  • CRISPR based regulatory devices provides a transcriptional framework to generate a library of “composable” regulatory devices.
  • The regulatory devices can be generated rapidly and the circuits have fast temporal response.