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

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in 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 rate 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 m gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34{degrees}C for 24 hours resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34{degrees}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.