Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

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.

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…The fatty acid composition has a significant impact on human health. In the emerging oil seed plant Camelina sativa , the FAD2 gene was knocked out thereby increasing the fraction of monounsaturated oleic acid to over 50% at the cost of less desirable polyunsaturated fatty acids (Jiang et al ; Morineau et al ). This approach is another example where the power of Cas9 technology was essential, because three homeoalleles of the hexaploid plant species had to be mutated.…”

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy

“…Furthermore, once a more efficient tissue culture system is obtained, one of our future goals is to estimate the genome editing efficiency of the TragCRISPR system by calculating the ratio of regenerated plants containing PDS mutations to all transgenic plants. This method of estimating efficiency is similar to what has been used in other CRISPR/Cas9 studies in various plant systems (Andersson et al, ; Braatz et al, ; Liu et al, ; Morineau et al, ; Wang et al, ).…”

“…In allohexaploid C. sativa , transformed with a construct containing one sgRNA, 26% of primary generation (T0) transgenic plants contained at least one mutated allele of the delta‐12‐desaturase gene ( CsFAD2 ) (Morineau et al, ). In the third generation of transgenic Camelina (T2), 98.4% of plants contained at least one mutated allele and 2.3% of transgenic lines showed mutations of all six alleles (Morineau et al, ). CRISPR/Cas9 was also applied in allohexaploid T. aestivum (wheat) to knock out the mildew‐resistance locus gene ( TaMLO ), and one sgRNA specifically targeting homoeologs from the A subgenome ( TaMLO‐A1 ) was designed (Wang et al, ).…”

“…Considering the polyploid nature of most crops and biofuel plants, the practicality of using CRISPR/Cas9 in polyploids is of great importance to plant breeders. With its simplicity and high efficiency, CRISPR/Cas9‐mediated genome editing has been reported in several polyploid crops (Weeks, ), including Triticum aestivum (6 x ) (Liang et al, ; Shan et al, ), Gossypium hirsutum (4 x ) (Li, Unver, & Zhang, ), Camelina sativa (6 x ) (Jiang et al, ; Morineau et al, ), Solanum tuberosum (4 x ) (Andersson et al, ; Butler, Atkins, Voytas, & Douches, ), Brassica napus (4 x ) (Braatz et al, ) , Nicotiana tabacum (4 x ) (Gao et al, ) and Panicum virgatum (4 x ) (Liu et al, ; Park et al, ). In these studies, simultaneous mutations of all alleles of target genes were obtained in the primary (T0) and/or the following generations of transgenic plants, which were frequently followed by phenotypic studies of the mutants.…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…For example, the labour and space-consuming production of immature embryos necessary for maize classical transformation could be replaced by alternative target tissues, like embryo slices from mature seed or leaf segments from seedlings whose preparation can be automated [21]. To overcome the constraints of plant regeneration (complexity and duration of the protocols, exposure to somaclonal variation), in planta transformation methods, independent from in vitro culture, are also being developed using whole plants or seeds [23][24][25]. However, these technologies are still restricted to very few plant species.…”