Abstract
CRISPR/Cas9 genome editing is a transformative technology that will facilitate the development of crops to meet future demands. However, application of gene editing is hindered by the long life cycle of many crop species and because desired genotypes generally require multiple generations to achieve. Single-celled microspores are haploid cells that can develop into double haploid plants and have been widely used as a breeding tool to generate homozygous plants within a generation. In this study, we combined the CRISPR/Cas9 system with microspore technology and developed an optimized haploid mutagenesis system to induce genetic modifications in the wheat genome. We investigated a number of factors that may affect the delivery of CRISPR/Cas9 reagents into microspores and found that electroporation of a minimum of 75,000 cells using 10–20 µg DNA and a pulsing voltage of 500 V is optimal for microspore transfection using the Neon transfection system. Using multiple Cas9 and sgRNA constructs, we present evidence for the seamless introduction of targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox2 and TaUbiL1. This study demonstrates the value and feasibility of combining microspore technology and CRISPR/Cas9-based gene editing for trait discovery and improvement in plants.
Highlights
Wheat is a primary staple food crop providing 20% of the calorie and protein intake for the global population
We successfully demonstrate the potential of microspores as explants for CRISPR/Cas9-based haploid mutagenesis by inducing targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox[2] and TaUbiL1
The Neon system is widely used for mammalian cell transfection, it had not been previously applied to plant cell transformation
Summary
Wheat is a primary staple food crop providing 20% of the calorie and protein intake for the global population. CRISPR/Cas[9] can generate targeted gene knock-outs and replacements which are invaluable for understanding the function of genes This innovative technology offers an efficient approach for genetic manipulation of crops without the retention of large transgene sequences in the final plant variety[8,9,10]. CRISPR/Cas[9] has been demonstrated in wheat[11,12,13,14,15], deployment of this technology in crop development is limited by the amount of time and resources required to produce modified homozygous genotypes through the conventional plant transformation based gene editing methods. We successfully demonstrate the potential of microspores as explants for CRISPR/Cas9-based haploid mutagenesis by inducing targeted modifications in an exogenous DsRed gene and two endogenous wheat genes, including TaLox[2] and TaUbiL1
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