Targeted genome editing with CRISPR/Cas9: a step towards generating nutritionally rich soybean

Abstract

Phytic acid is 75-80% of the total phosphorus in cereal and legume seeds but maximum portion of it is not accessible as the monogastric animals don't have phytase in their digestive tracts which is important for hydrolysis of the phytate molecule. This leads to eutrophication due to the influx of phosphorus from undigested animal waste which is the major source of agricultural phosphorus runoff. Also the polyanionic antinutrient, phytic acid binds to mineral cations and makes them unavailable for absorption by animals leading to mineral deficiencies. Low phytate soybean is thus desirable not only for improving human health but also for reducing the environmental phosphorus load. The implications of phytic acid on the environment, human nutrition and livestock industry have sparked numerous nutritional studies focussing on reduction of its levels. Unfortunately, the negative and altered phenotypes obtained by classical breeding mutations and reverse genetics approaches necessitate the usage of a more targeted and efficient biotechnological intervention for tissue-specific knockdown of phytic acid, obliterating the negative impacts on basal metabolic functions. The steps undertaken in the present study are for developing low-phytate soybean using the latest developed, targeted genome editing tool, CRISPR/Cas9 system for precise and efficient editing of the IPK2 gene of phytic acid biosynthesis pathway. A CRISPR/Cas9 construct for editing the IPK2 gene, involved in phytic acid biosynthesis was successfully designed and generated.