Scientists have successfully used CRISPR/Cas9 gene scissors to reduce the content of the free amino acid asparagine in wheat. Free asparagine is present in higher concentrations in wheat grain. Acrylamide can be formed from this free asparagine, together with reducing sugars, when wheat products are heated. Acrylamide has been shown to have carcinogenic properties.
In the study, CRISPR/Cas9 was used to knock out all the alleles of the asparagine synthetase 2 gene (ASN2 gene) in wheat, which is mainly expressed in the grain. Wheat has four other ASN genes, but the ASN2 gene is mainly expressed in the grain. The aim was to reduce the amount of free asparagine in the grain, while still keeping sufficient asparagine for protein biosynthesis available in other parts of the grain. All ASN genes occur as single gene copies in the 3 subgenomes of wheat. This means that the ASN2 gene occurs a total of six times in the wheat genome.
The various genome-edited wheat lines that were produced showed a greatly reduced asparagine concentration in the grain. In some second generation genome-edited wheat plants, the asparagine content was reduced by 90% compared to the wild type. Such a strong reduction of the asparagine content in wheat grain has not previously been achieved using other methods.
Interestingly, however, some of these plants were found to hardly germinated at all. Apparently, the amount of asparagine formed was not sufficient for this to occur. Asparagine is an amino acid that is needed in protein biosynthesis, which could explain the observed effect. The scientists circumvented this problem by spraying a certain amount of asparagine dissolved in water onto the plants. The genome-edited wheat plants subsequently germinated.
Surprisingly, the scientists also found that the asparagine concentration can fluctuate from generation to generation. This is most likely due to the fact that another of the total of five ASN genes is produced more with increased temperatures, and this then leads to an increase in asparagine content in the grain. The scientists explained that the greenhouse was exposed to increased temperatures when the wheat plants were grown, and that this is very likely the reason for the fluctuations.
The genome-edited wheat plants are to be tested in field trials.
The genome-edited wheat plants will now be tested in field trials together with wheat plants that have been generated using chemical mutagens that have a reduced asparagine content compared to the wild type. However, wheat lines bred from chemical mutagenesis show a higher asparagine content than the wheat modified with CRISPR/Cas9. In direct comparison, the wheat lines from induced mutagenesis and from the CRISPR/Cas experiments differ according to the position of the genetic modification and the number of modifications: the scientists can use the gene scissors to determine exactly which DNA sequence(s) are changed and select the appropriate genetic changes. In addition, several alleles of the ASN2 gene can be changed simultaneously, which is difficult to achieve with induced mutagenesis, especially in polyploid plants such as wheat.
The trials will show whether, and to what extent, the asparagine content in the grain of wheat plants fluctuates in changing environmental conditions. The field trials are scheduled to start in autumn 2021 and will cover an area of 1500 square metres.
Raffan, S.; Sparks, C.; Huttly, A.; Hyde, L.; Martignago, D.; Mead, A.; Hanley, S.J.; Wilkinson, P.A.; Barker, G.; Edwards, K.J.; et al. Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2. Plant Biotechnology Journal 2021, 19, 1602-1613, doi: https://doi.org/10.1111/pbi.13573.