CRISPR-Cas9 Mediated Biofortification of Local Wheat through Disruption of IPK1 Gene for Folate and Micronutrient Bioavailability

Abstract

Biofortification of cereal grains with improved iron and other micronutrients is a useful strategy to combat malnutrition. Phytic acid (PA) is an important anti-nutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, thus creating malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis. IPK1 gene is the member of IPK superfamily of enzymes. These enzymes are involved in the synthesis of Inositol polyphosphates which act as second messengers to perform vital functions in eukaryotic cells. The present study identified and classified 28 candidate wheat inositol phosphate multikinase genes (IPMKs), members of IPK superfamily, into three groups (IPPK1, IPPMK2, and ITPK). These IPMK groups were defined on the basis of conserved domains, chromosome locations and gene structures. On the basis of analysis of the phylogenetic relationships among wheat IPMKs, eight pairs of paralogous IPMKs were identified. All the 28 TaIPMKs exhibited diverse expression patterns in different tissues at different developmental stages showing diversity in their biological functions. Biofortification of cereal grains with iron and other micronutrients through genetic manipulation is a desirable strategy and plausible solution to mitigate malnutrition in developing countries. But earlier efforts to suppress the expression of inositol pentakisphosphate 2- kinase 1 (IPK1) gene -codes for an enzyme involved in the final step of phytate biosynthesis which is an anti-nutrient chelating agent- remained fragmented. In this study, 3 TaIPK1 homeologs designated as TaIPK1.A, TaIPK1.B, and TaIPK1.D from wheat corresponding to A, B and D genomes respectively were isolated. The expression of TaIPK1.A transcripts was stronger in early stages of grain filling in both the selected wheat varieties namely “Pakistan 2013” and “Borlaug 2016”. In order to disrupt the TaIPK1.A homeolog in Borlaug 2016 two guide RNAs targeting the 1st and 2nd exons in CRISPR vectors were annealed and shuttled to Borlaug 2016 cultivar. Targeting of TaIPK1.A led to Cas9 induced mutations in the first generation plants at frequencies of 12.7% and 10.8%, respectively. Plants carrying frame-shifted mutagenesis could be recovered in T1 generation. Sequencing analysis revealed deletion of 1 to 23 nucleotides and even an addition of 1 nucleotide in various genome-edited lines. Variable frequencies of allele 1 and allele 2 were observed for different mutant lines. Remarkably, alterations in spikelet number were recorded in 3 GE plant mutant lines Abstract CRISPR-Cas9 Mediated Biofortification of Local Wheat through Disruption of IPK1 Gene for Folate and Micronutrient Bioavailability xv T2-L4, T2-L9 and T2-L13. A nutritional phenotypic screen identified the maximum reduction in the phytic acid content for T2-L9M2_E genome-edited plants (0.44ug/g) compared to control plants (1.60ug/g). The other two GE lines i.e. T2-L4M1_A and T2-L13M3_F also showed significant reduction in PA content as 1.22 and 1.12ug/g, respectively. Further, micronutrient accumulation of Fe and Zn in grains showed a significant (at P<0.01) increase in genome edited lines of TaIPK1 compared to control seeds. Maximum Fe content (83.61ug/g) were observed in T2-L9M2_E line plants, while higher Zn content (64.23ug/g) were demonstrated in T2-L13M3_F genome edited line plants. This study demonstrates the implications of CRISPR-Cas9 in generating knockouts in wheat for IPK1 gene. These studies have potential application in fostering efforts to fortify wheat grains with Fe and Zn for resource poor in developing countries.