Assistant Professor University of Tennessee Knoxville, Tennessee
Body of Abstract: The prospect of designing complex genetic circuits for precise modulation of gene expression has the potential to revolutionize agriculture. Among the synthetic biology toolbox for engineering plants, dead Cas9 (dCas9) is a catalytically inactive Cas9 mutant without endonuclease activity that still conserves intact ability to bind the DNA target complementary to a sgRNA. Recently, dCas9 has been used to design synthetic transcription factors (TFs) for modulating the activity of promoter regions. To improve the plant synthetic biology toolbox, in this work, a library of sgRNA/dCas9TF combinations for modulating the activity of twelve promoters functional in plants, covering a range of activities from low (nos promoter) to high (CaMV 35S promoter) expression level were tested in leaf. Three activation domains (VP16, VP64 and VP128) from the human alpha herpesvirus-1 (HSV-1), the TV domain (VP128-TAL) along with a novel activation domain from the QF TF from Neurospora crassa were tested. In parallel, the plant-specific SRDX domain was used to test repression of the same plant promoters. From the first screening, sgRNA/dCas9TF combinations were down selected for efficient modulation of transgene expression in plant cells. As a valuable tool, the low-activity nos promoter can be activated ~ 8-fold using a single sgRNA/dCas9TF combination. We anticipate that in the near future, these novel tools will find valuable applications in agriculture, including the design of precise on/off genetic switches for sensing and reporting environmental stimuli.
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 (DISTAR Case).