Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Enabling Synthetic Biology in Setaria and Sorghum Through Targeted Mutagenesis and Programmed Transcriptional Regulation

Authors:

Erik Myers1* (myers730@umn.edu), Janina Tamborski2, Hui Jiang3, Britney Millman3, Ashley Snouffer3, Nathan Butler1, Albert Kausch4, Jennifer Brophy2, Daniel Voytas1, Ivan Baxter3

Institutions:

1University of Minnesota–St. Paul; 2Stanford University–Palo Alto; 3Donald Danforth Plant Science Center; 4University of Rhode Island–Kingston

URLs:

Goals

Bioenergy feedstocks need to be deployed on marginal soils with minimal inputs to be economically viable and have a low environmental impact. Currently, crop water supply is a key limitation to production. The yields of C4 bioenergy crops such as Sorghum bicolor have increased through breeding and improved agronomy. Still, the amount of biomass produced for a given amount of water use (water-use efficiency, or WUE) remains unchanged. Therefore, this project aims to develop novel technologies and methodologies to redesign the bioenergy feedstock sorghum for optimal WUE. Within this broader context, this subproject is using Setaria viridis as a rapid-cycling model for gene discovery. Researchers aim to develop and demonstrate novel methods and resources to accelerate both the production of genetic variants and phenotyping of WUE traits as part of reverse and forward genetics approaches to discover genes regulating stomatal patterning and WUE.

Abstract

Improving WUE in Sorghum requires the ability to manipulate endogenous genes and gene expression patterns. Researchers are implementing several technologies for the genetic improvement of sorghum as well as the model C4 plant, Setaria. In both species, the delivery of DNA to cells is critical to alter the genetic code. Researchers currently have a robust pipeline for Setaria transformation; however, traditional methods of Sorghum transformation are laborious and time consuming. To expedite Sorghum transformation, researchers are using developmental regulators, including BABY BOOM and WUSCHEL2, to promote somatic embryogenesis from transformed sorghum leaf cells. Using traditional transformation methods in Setaria, researchers have succeeded in making specific nucleotide substitutions through prime editing. This technology enables precise small insertions (~30) or deletions (~100bp). Researchers are currently focused on introducing changes that would alter the kinetic activity of the key photosynthetic enzyme, PEP Carboxylase. To accelerate gene editing, researchers are using RNA viruses to deliver gene editing reagents through infection. Because the cargo capacity of most plant RNA viruses is limited to approximately 1 kb, researchers have made transgenic lines of sorghum and Seteria that express Cas9; sgRNAs are expressed from the virus, and gene editing occurs through infection. The current goal is to use viruses to edit the germline so that seeds can be harvested with heritable modifications to their genomes. To achieve precise control over gene expression, researchers are building synthetic genetic circuits to enable spatial and tissue-specific control over gene expression. Synthetic circuits offer a means to reprogram plant development and control growth. Finally, to expedite classical genetics, researchers have generated a male sterile line of Setaria using CRISPR/Cas9 by making targeted, inactivating mutations in a gene important for pollen development. These male sterile lines are currently being tested and promise to greatly accelerate genetic analyses in Setaria.

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE-SC0023160 and DE-SC0018277.