Steps Towards CRISPR-Based Process Control in a Cell-Free System

Maxwell, C., North Carolina State University
Beisel, C. L., North Carolina State University
Lucks, J. B., Cornell University
Noireaux, V., University of Minnesota
Cell-free systems (TXTL) have the potential to allow manufacturing of biologicals--including antimicrobials and vaccines--outside of laboratory settings. However, effective process control is needed to ensure that the appropriate amount of a product is produced by these systems despite environmental variability. RNA sequestration could allow the error computation needed for proportional control of output, but this has not been demonstrated. We use a TXTL system to prototype and tune the components of a CRISPR/Cas9 proportional controller in a cell-free system. We demonstrate error computation based on antisense sequestration of a repressing crRNA. Robust proportional control relies on the ability of the system to increase output when it is below the set point. We demonstrate a novel approach to converting catalytically dead Cas9 (dCas9) into a transcriptional activator with greater than ten-fold activation of gene expression. Our results lay the foundations for process control in cell-free systems and possibly in living cells.