12/28/2022
Transformation and Gene Editing in the Bioenergy Grass Miscanthus
First demonstration of targeted editing using the CRISPR/Cas9 system
Leaf phenotypes in five Miscanthus genotypes transformed with the gene editing vector pHA194. (WT = isogenic wild-type line, lw1 = transgenic edited shoots/plantlets/plants.)
[Reprinted under a Creative Commons license (CC BY 4.0) from Trieu, A., et al. 2022. DOI:10.1186/s13068-022-02241-8]
The Science
Miscanthus, a C4 carbon fixation plant genus in the Poaceae family, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. To date, biotechnology-based procedures to genetically improve Miscanthus species have only included plant transformation procedures for introducing exogenous genes into the host genome at random, non-targeted sites. Research by the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) now demonstrates, for the first time, that the genomes of three Miscanthus species can be edited via the CRISPR/Cas9 system.
The Impact
The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.
Summary
CABBI researchers developed procedures for gene editing via CRISPR/Cas9 in Miscanthus species, including five genotypes representing three species. Prior research in Zea mays had demonstrated that the lemon white1 (lw1) gene is a promising target for visual confirmation of gene editing in other species.
Using sequence information from both miscanthus and sorghum, researchers identified lw1 orthologs. Embryogenic calli of Miscanthus sacchariflorus, M. sinensis, and M. x giganteus were transformed via either particle bombardment (biolistics) or Agrobacterium tumefaciens, introducing the Cas9 gene and three guide RNAs (gRNAs) to edit lw1. The designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles) and was confirmed by Sanger sequencing.
Principal Investigator
Nancy Reichert
Mississippi State University
[email protected]
BER Program Manager
Shing Kwok
U.S. Department of Energy, Biological and Environmental Research (SC-33)
Biological Systems Science Division
[email protected]
Funding
This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Biological and Environmental Research program under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy.
References
Trieu, A., et al. 2022. “Transformation and Gene Editing in the Bioenergy Grass Miscanthus,” Biotechnology for Biofuels and Bioproducts 15, 148. DOI:10.1186/s13068-022-02241-8.