Targeted mutagenesis in wheat microspores using CRISPR/Cas9

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.

Wheat Blast in Bangladesh: The Current Situation and Future Impacts

“…However, mutagenesis of wheat using CRISPR/Cas9 could be a faster approach for development of novel blast resistant wheat. To ease the complication in transformation of wheat, a new targeted mutagenesis in wheat microspores using CRISPR/ Cas9 has recently been established ( Bhowmik et al, 2018 ). A strategy of introducing blast-resistance genes from wild wheat progenitors proposed by Wulff and Dhugga (2018) could also be helpful for rapid development of elite cultivars to confer multilayered disease resistance.…”

Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges

“…On the other hand, haploid microspores, or immature pollen grains can be easily isolated from many different species in a relatively short period of time, and used as targets for transformation and early screening of gene editing events. Although a functional microspore-based gene editing system using electroporation, biolistic and Agrobacterium -mediated delivery has several advantages compared to conventional plant transformation-based systems, it has very low efficiency regarding regeneration of microspore edited plants (Brew-Appiah et al, 2013 ; Bhowmik et al, 2018 ).…”

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?

“…F0 animals generated by CRISPR/Cas9 technology are often mosaic; to screen for the presence of insertion/deletion mutations from a large number of F0 animals, a variety of methods can be used other than Sanger sequencing. The easiest methods are to take screening into consideration when selecting target sites, such that the target site contains a restriction enzyme site that will be lost along with the lesion; this loss can be identified through PCR/restriction enzyme analysis (Gao et al, 2015;Bhowmik et al, 2018). Alternatively, segmental deletions can be generated by simultaneously targeting multiple sites.…”