Transformation and gene editing in the bioenergy grass Miscanthus

Biotechnology for Biofuels and Bioproducts

Research

Thomas E. Clemente¹  Rebecca L. Billingsley²  Nancy A. Reichert²  Anjali Arpan²  Erik J. Sacks³  Stephen P. Moose³  Shilpa Manjunatha⁴  Yokshitha Bathula⁴  Rebekah Wood⁴  Kankshita Swaminathan⁴  Pradeepa Hirannaiah⁴  Mohammad B. Belaffif⁴  Anthony Trieu⁴ 

[1] Department of Agronomy and Horticulture, University of Nebraska-Lincoln, 68583, Lincoln, NE, USA;DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois Urbana-Champaign, 61801, Urbana, IL, USA;Department of Biological Sciences, Mississippi State University, 295 Lee Blvd., 39762, Mississippi State, MS, USA;DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois Urbana-Champaign, 61801, Urbana, IL, USA;Department of Crop Sciences, E.R. Madigan Laboratory, University of Illinois Urbana-Champaign, 1201 W. Gregory Dr., 61801, Urbana, IL, USA;DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois Urbana-Champaign, 61801, Urbana, IL, USA;HudsonAlpha Institute for Biotechnology, 601 Genome Way, 35806, Huntsville, AL, USA;DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois Urbana-Champaign, 61801, Urbana, IL, USA;

关键词: Miscanthus sinensis;    Miscanthus sacchariflorus;    Miscanthus;    Transformation;    CRISPR/Cas9;    Gene editing;    Lemon white1;   

DOI  :  10.1186/s13068-022-02241-8

 received in 2022-06-29, accepted in 2022-12-08,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundMiscanthus, 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. sacchariflorus 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.ResultsWe 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. sacchariflorus, 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.ConclusionsWe 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 editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.Graphical Abstract

【 授权许可】

CC BY   
© The Author(s) 2022

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