Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics

Abstract

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in the ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation efficiency bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 μm gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34 °C for 24 h resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34 °C treatment produced two M₀ mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.

Keywords: albino phenotype; biolistics; genome editing; high temperature; microparticle size; phytoene desaturase gene (PDS); rupture disk pressure; transformation; wheat.

Figure 1

Schematic diagrams of three transformation vectors used for wheat transformation and gene editing. (A) pAct1IHPT-4 is a 5870-bp plasmid containing hygromycin phosphotransferase (HPT) driven by the OsAct1 promoter and its intron. (B) pAct1IDsRED is a 5139-bp plasmid containing DsRED driven by the OsAct1 promoter and its intron. (C) pRGE-PDS-PS2 is a 10,122-bp plasmid containing 2 gene cassettes where gRNA is driven by the Tau6 promoter and Cas9 is driven by the OsUbi10 promoter.

Figure 2

Stable wheat transformation via biolistics: (A) Harvest immature wheat spikes 10–14 days post-anthesis; (B) isolate immature embryos sized 1.7–2.2 mm and place on osmoticum medium for 4 h; (C) shoot gold particles using gene gun with desired gold particle size and rupture disc pressure; (D) plant tissue is subjected to three rounds of callus induction media containing selection, subculturing every 3 weeks; (E) larger callus pieces derived from a single immature embryos are broken up and placed on regeneration medium for shoot formation; (F) plantlets that are at least 1 cm in height are transferred to rooting medium in Phytatrays and grown to size until they can be transferred to soil.

Figure 3

DsRED expression in different tissue types in transgenic wheat. (A) Transient DsRED expression and brightfield of an immature wheat embryo 3 days post-bombardment. (B) The formation of a DsRED sector growing 6–8 weeks post-bombardment compared with brightfield image of the same sector. (C) A stable transformation showing DsRED expression in leaf tissue in comparison to a wild-type control. (D) A stably transformed DsRED plantlet 10–12 weeks post-bombardment under fluorescence and brightfield.

Figure 4

Volume (weight) and surface area of different gold particle sizes. Different gold particles are capable of holding different quantities of DNA. (A) The diameter of a sphere directly affects the number of gold particles by weight. As diameter increases, the number of gold particles in a fixed weight decreases. The smaller the gold particle, the more particles will be available to be coated in DNA for each bombardment prep. (B) Larger diameter directly affects surface area of each particle. The larger the diameter, the greater the surface area. The difference in diameter between 0.4 μm to 1.0 μm results in a 6.25-fold increase in surface area. This means that larger particles are capable of holding a greater amount of DNA.

Figure 5

Triple biallelic knockout mutants showing albino phenotype. Photos depicting albino PDS triple biallelic mutants in plates, as well as in Phytatrays. Both albino events #1 and #2 produced as a result of the 34 °C 1-day heat treatment, shown in plate and Phytatray.

Figure 6

Phenotype of M₁ progeny plants derived from event PC14A with monoallelic and biallelic mutations in the three different genomes. M₁ progeny segregation from PC14A which contained two monoallelic mutations and one biallelic mutation. Photo of two albino M₁ progeny of the twenty-eight total plantlets (red arrows).