A highly efficient CRISPR-Cas9-based gene editing system in oat (Avena sativa)

Abstract

Cultivated oat (Avena sativa) is an emerging cereal for healthy lives owing to its unique characteristics, such as high {beta}-glucan and oil content, distinctive fatty acid composition, and gluten-free nature. The recent unravelling of the 12.5 Gb hexaploid oat genome underlined breeding barriers caused by ancestral translocations and inversions, leading to recombination suppression and pseudo-linkage further hindering conventional trait introgression. Over the past decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has been extensively used for crop improvement and functional genomics in all other cereals except oats. Its large repetitive genome with three sub-genomes, lack of efficient transformation, recalcitrant nature, and complex molecular screening due to gene redundancy have been major obstacles to gene editing success in oat. We report the first successful CRISPR-Cas9-based gene editing in oat in three genes -- AsTLP8, AsVRN3 and AsVRN3D with gene-editing efficiency of up to 41.1%. The gene-edited plants for all the genes carried deletions and/or one base insertion. Further analysis of VRN3 T1 and T2 mutants revealed bent leaves in heterozygous knockouts (AACCdD), while an extended vegetative growth phase was seen in the T1 homozygous and biallelic mutants (aaccdd), accentuating the important role of VRN3 in oat development. We are confident that this highly efficient oat gene editing system will pave the way for a deeper molecular understanding of this healthy cereal, deciphering oats functional genomics, and creating genetic diversity at the cold spots of recombination in oat.