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.

Figure 1

Effect of pulsing voltage on delivery of Cas9 and DsRed expression construct into wheat microspores using the Neon electroporation system. Representative images of fluorescein diacetate (FDA) stained viable and DsRed expressing wheat microspores after 48 h after electroporation are shown. The highest microspore survival and transfection efficiency were observed at 500 V (1.6% transfection efficiency, 50% cell viability).

Figure 2

Optimization of electroporation parameters for the delivery of Cas9 and DsRed expression construct into wheat microspores. The effect of buffer composition (A), DNA concentration (B) and microspore density (C) on transfection efficiency is shown. The microspore transfection using optimised electroporation parameters (D) resulted in the highest transfection efficiency of 2.2% (E), as measured by flow cytometry. The error bars represent standard error of the mean (SEM).

Figure 3

Microspore-derived homozygous doubled haploid plants. Epifluorescent microscopy images showing germinating embryos and green shoots regenerated from wild type (control) and transformed microspores (transformants) expressing DsRed. Scale bar, 1000 µM.

Figure 4

CRISPR/Cas9-based editing of the exogenous DsRed reporter gene in wheat microspores. (A)Fluorescence emitted from DsRed produced in microspores co-transfected with the DsRed_Cas9 and gDsRed-2 constructs (right panel) compared to control cells expressing only DsRed_Cas9 construct (Left panel). (B)Mutations detected at or near the cleavage site in sequenced products of DsRed from microspores co-transfected with the DsRed_Cas9 and gDsRed-2 constructs.

Figure 5

CRISPR/Cas9-based editing of the endogenous TaLox2 gene in wheat microspores. (A) Schematic diagram showing the pJIT163-2NLSCas9/gTaLox2-target3 construct used for mutagenesis of TaLox2 gene in wheat microspores. (B) PCR/restriction enzyme assay to detect mutations in the TaLox2 gene induced by Cas9/ gTaLox2. (C) A 2 bp deletion detected at the cleavage site in the sequenced product of TaLox2 from microspores transfected with pJIT163-2NLSCas9/gTaLox2-target3 construct. Sanger sequencing electropherograms are shown below.

Figure 6

CRISPR/Cas9-based editing of an endogenous ubiquitin gene in wheat microspores. (A) Schematic diagram of the pEEE005-NLSCas9/gUbi1 and pEEE006-NLSCas9/gUbi1/GFP constructs used for mutagenesis of in wheat microspores. (B) PCR/restriction enzyme assay to detect mutations in TaUbiL1 induced by Cas9/ gUbi1. (C) Deletions detected at the cleavage site in sequenced products of all three homoeologs of TaUbiL1 from wheat microspores transfected with pEEE005-NLSCas9/gUbi1 or pEEE006-NLSCas9/gUbi1/GFP constructs. The three homoeologues of TaUbiL1 were defined based on multiple single nucleotide polymorphisms in each background (sequence not shown) for the three genomes in this locus. (D) Sanger sequencing electropherograms showing mutations in TaUbiL1 homoeologues from the A, B and D genomes of wheat.