Body of Abstract: Plant synthetic biology relies on targeted genomic regulation to engineer complex genetic circuits for the purposes of crop improvement or the installation of other valuable pathways. CRISPRa and CRISPRi are strategies of targeted regulation that use the catalytically dead variant of the Cas9 nuclease (dCas9). Specifically, dCas9, either alone or fused to a transcriptional activation or repression domain, is guided by small RNAs (gRNAs) to target sequences in a promoter, thereby regulating promoter activity and respectively activating or blocking transcription of target genes. In previous studies in other organisms, the use of a single gRNA sequence has not resulted in highly effective transcriptional control, but it has been shown that multiplexing guides, by targeting multiple gRNAs to a single genetic locus, can enhance the efficiency. When multiple gRNAs are used, however, their independent activities may interact to either have a synergistic effect on transcriptional control or to interfere with one another. We present here a simulation-based analysis of the potential for transcriptional regulation using CRISPRi with both single and multiplexed gRNA guides. We have evaluated transcriptional repression with a varying number of gRNAs in simulation with respect to tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation, and use the results to produce recommendations for the engineering of CRISPRi plant transcriptional regulation systems.
This work was supported by DARPA contract HR0011-18-2-0049. This document does not contain technology or technical data controlled under either U.S. International Traffic in Arms Regulation or U.S. Export Administration Regulations. Views, opinions, and/or findings expressed are those of the author(s) and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. Approved for public release, distribution unlimited.