Transformation and gene editing in the bioenergy grass Miscanthus

Authors

Anthony Trieu, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign
Mohammad B. Belaff, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign
Pradeepa Hirannaiah, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign
Shilpa Manjunatha, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign
Rebekah Wood, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign
Yokshitha Bathula, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign
Rebecca L. Billingsley, Mississippi State University, University of Illinois Urbana-Champaign, Urbana
Anjali Arpan, Mississippi State University, University of Illinois Urbana-Champaign, Urbana
Erik J. Sacks, University of Illinois Urbana-Champaign
Thomas E. Clemente, University of Nebraska - Lincoln, University of Illinois Urbana-ChampaignFollow
Stephen P. Moose, University of Illinois Urbana-Champaign
Nancy A. Reichert, Mississippi State University, University of Illinois Urbana-Champaign, Urbana
Kankshita Swaminathan, HudsonAlpha Institute for Biotechnology, University of Illinois Urbana-Champaign

Date of this Version

12-28-2022

Citation

Trieu et al. Biotechnology for Biofuels and Bioproducts (2022) 15:148 https://doi.org/10.1186/s13068-022-02241

Comments

Open access.

Abstract

Background: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariforus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites.

Results: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariforus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors.

Conclusions: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good