Selective gene dosage by <scp>CRISPR</scp>‐Cas9 genome editing in hexaploid <i>Camelina sativa</i>

TLDR

Gene dosage drives phenotypic variation in plants, and the hexaploid oilseed Camelina sativa, with three closely related subgenomes, offers an ideal system to explore combinatorial mutant creation. The study aims to engineer gene dosage in polyploid genomes to create a versatile tool for plant breeding. CRISPR‑Cas9 was used to selectively mutate the three CsFAD2 genes, reducing polyunsaturated fatty acids and boosting oleic acid content. Across four generations, a diverse array of heritable CsFAD2 mutations—including single, double, and triple loss‑of‑function alleles—was generated, producing lines with 10–62 % oleic acid that demonstrate the critical role of delta‑12 desaturation and provide a rich resource for breeding.

Abstract

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta-12-desaturase (FAD2) genes was achieved by CRISPR-Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss-of-function mutants revealed the importance of delta-12-desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.