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Title: Transformation and gene editing in the bioenergy grass Miscanthus

Journal Article · · Biotechnology for Biofuels and Bioproducts
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  1. HudsonAlpha Inst. for Biotechnology, Huntsville, AL (United States)
  2. Mississippi State Univ., Mississippi State, MS (United States)
  3. Univ. of Illinois at Urbana-Champaign, IL (United States)
  4. Univ. of Nebraska, Lincoln, NE (United States)

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. 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. 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. 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. 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 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.

Research Organization:
Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
SC0018420
OSTI ID:
1914910
Journal Information:
Biotechnology for Biofuels and Bioproducts, Vol. 15, Issue 1; ISSN 2731-3654
Publisher:
BioMed CentralCopyright Statement
Country of Publication:
United States
Language:
English

References (46)

Data Mining by Pluralistic Approach on CRISPR Gene Editing in Plants journal July 2019
Progress in Optimization of Agrobacterium-Mediated Transformation in Sorghum (Sorghum bicolor) journal September 2018
Map-Based Cloning of zb7 Encoding an IPP and DMAPP Synthase in the MEP Pathway of Maize journal September 2012
Targeted mutagenesis in rice using CRISPR-Cas system journal September 2013
Optimised expression cassettes of hpt marker gene for biolistic transformation of Miscanthus sacchariflorus journal August 2019
Nomenclature of Miscanthus xgiganteus (Poaceae) journal January 2001
Direct and indirect in vitro regeneration of Miscanthus × giganteus cultivar Freedom: effects of explant type and medium on regeneration efficiency journal March 2015
A global yield dataset for major lignocellulosic bioenergy crops based on field measurements journal August 2018
Application of the CRISPR–Cas System for Efficient Genome Engineering in Plants journal November 2013
Establishment of an efficient in vitro culture and particle bombardment-mediated transformation systems in Miscanthus sinensis Anderss., a potential bioenergy crop: IN VITRO CULTURE AND TRANSFORMATION journal January 2011
Genome biology of the paleotetraploid perennial biomass crop Miscanthus journal October 2020
Agrobacterium-mediated genetic transformation of Miscanthus sinensis journal December 2013
A framework genetic map for Miscanthus sinensis from RNAseq-based markers shows recent tetraploidy journal January 2012
Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System journal January 2016
NewAgrobacterium helper plasmids for gene transfer to plants journal July 1993
Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars journal July 2019
A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures journal July 1962
Recent developments in genome editing and applications in plant breeding journal October 2017
Miscanthus : a case study for the utilization of natural genetic variation journal November 2014
Stress-Tolerant Feedstocks for Sustainable Bioenergy Production on Marginal Land journal February 2015
Problems and possibilities of monocot transformation journal March 2011
Mfold web server for nucleic acid folding and hybridization prediction journal July 2003
CRISPR for Crop Improvement: An Update Review journal July 2018
Warm-Season Grass Monocultures and Mixtures for Sustainable Bioenergy Feedstock Production in the Midwest, USA journal November 2018
Phenotypic Characterization of Transgenic Miscanthus sinensis Plants Overexpressing Arabidopsis Phytochrome B journal January 2014
Efficient genome editing in plants using a CRISPR/Cas system journal August 2013
Biotechnology of Miscanthus book January 2013
Evaluation of Selectable Markers for Obtaining Stable Transformants in the Gramineae journal February 1988
A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants journal August 2015
High Resolution Genetic Mapping by Genome Sequencing Reveals Genome Duplication and Tetraploid Genetic Structure of the Diploid Miscanthus sinensis journal March 2012
Environmental costs and benefits of growing Miscanthus for bioenergy in the UK journal August 2015
Targeted genome modification of crop plants using a CRISPR-Cas system journal August 2013
Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation journal September 2014
In Vitro Propagation of Miscanthus sinensis journal October 1990
Establishment of Miscanthus sinensis with decreased lignin biosynthesis by Agrobacterium–mediated transformation using antisense COMT gene journal February 2018
Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage journal January 2022
A Study of Pleiotropic Genes in Maize journal April 1954
TransgenicMiscanthus lutarioripariusthat co-expresses theCry 2Aa#andBargenes journal December 2019
DNA targeting specificity of RNA-guided Cas9 nucleases journal July 2013
Developing a long-term and powerful in vitro culture and Agrobacterium-mediated transformation system for Miscanthus sinensis (Poaceae) journal March 2021
MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability journal January 2013
The use of dna sequencing (ITS and trnL-F), AFLP, and fluorescent in situ hybridization to study allopolyploid Miscanthus (Poaceae) journal February 2002
A Review on Miscanthus Biomass Production and Composition for Bioenergy Use: Genotypic and Environmental Variability and Implications for Breeding journal September 2014
OrthoFinder: phylogenetic orthology inference for comparative genomics journal November 2019
Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing journal February 2016
High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize journal April 2018