Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far, the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged a recent protocol for producing an E. coli-based cell-free expression system with two CRISPR-associated proteins, Csy4 and dCas9, expressed prior to harvest. We found that pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrated the benefits and versatility of dCas9 and Csy4 through the use of RNA circuitry based on a combination of single guide RNAs, small transcriptional activator RNAs, and toehold switches. For instance, we show that Csy4 processing increased 4-fold the dynamic range of a previously published AND-logic gate. Additionally, blending the CRISPRenhanced extracts enabled us to reduce leakage in a multiple inputs gate, and to extend the type of Boolean functions available for RNA-based circuits, such as NAND-logic. Finally, we reported the use of simultaneous transcriptional and translational reporters in our RNA-based circuits. In particular, the AND-gate mRNA and protein levels were able to be independently monitored in response to transcriptional and translational activators. We hope this work will facilitate the adoption of advanced processing tools for RNAbased circuit prototyping in a cell-free environment.

Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged the most recently published protocol for E. coli-based cell extracts with the endogenous production of two CRISPR-associated proteins, Csy4 and dCas9. We found pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrated the benefits and versatility of dCas9 and Csy4 through the use of RNA circuitry based on a combination of single-guide RNAs, small transcriptional activator RNAs, and toehold switches. For instance, we show that Csy4 processing increased fourfold the dynamic range of a previously published AND-logic gate. Additionally, blending the CRISPR-enhanced extracts enabled us to reduce leakage in a multiple inputs gate, and to extend the type of Boolean functions available for RNA-based circuits, such as NAND-logic. Finally, the use of dual transcriptional and translational reporters for the engineering of RNA-based circuits, allowed us to gain better insight into their underlying mechanisms. We hope this work will facilitate the adoption of advanced processing tools for RNA-based circuit prototyping in a cell-free environment.