Efficient Designing, Validation, and Transformation of GmIPK2 Specific CRISPR/Cas9 Construct for Low-Phytate Soybean
Joshna Jose1* and Archana Sachdev2
1Department of Biochemistry, ICAR- Indian Agricultural Research Institute, New Delhi,India
2Principal Scientist, Division of Biochemistry, ICAR-IARI, New Delhi, India
*Corresponding Author: Joshna Jose, Department of Biochemistry, ICAR- Indian Agricultural Research Institute, New Delhi, India.
January 27, 2022; Published: February 21, 2022
Background: The nutritive potential of soybean is limited by the presence of an absorption inhibitor – phytic acid. Development of low-phytate soybean has been envisioned; but, limited variations in the gene pool and the regulatory hurdles have handicapped this process. The present study uses type II CRISPR/Cas9 system for precise editing of GmIPK2 (inositol polyphosphate 6-/3-/5-kinase).
Methodology: Single guide RNA (sgRNA) sequences were designed and validated for their efficiency by using various webtools (CRISPR-scan, RNA fold server, and Cas-OFFinder). A single binary vector carrying the guide RNA and Cas9 cassette was designed and expressed transiently in soybean leaf discs by using AGRODATE (Agrobacterium-mediated Disc Assay for Transient Expression) method to edit the GmIPK2 gene. We observed deletions of 2 to 5 nucleotides in the target region of the analyzed leaf discs; thus, validating efficacy of the construct in vivo. Stable transformation of soybean with Cas9/gRNA-GmIPK2 construct was also carried out.
Conclusion: The experimental sensitivity resulting from sgRNA efficiency is a major hurdle in successful CRISPR/Cas9-based genome editing. Employing multiple webtools and use of transient expression assays as depicted in this study can speed-up the CRISPR/Cas9 based editing in recalcitrant crops like soybean.
Keywords: CRISPR/Cas9; Genome Editing; Soybean; Phytic Acid; Crop Improvement
- FAO. World Food and Agriculture - Statistical Yearbook 2020. Rome (2020).
- Krishnan V., et al. “Enhanced nutraceutical potential of gamma irradiated black soybean extracts”. Food chemistry 245 (2018): 246-253.
- Chitra U., et al. “Variability in phytic acid content and protein digestibility of grain legumes”. Plant Foods for Human Nutrition 47 (1995): 163-172.
- Kumari S., et al. “In vivo' bioavailability of essential minerals and phytase activity during soaking and germination in soybean ('Glycine max'L.)”. Australian Journal of Crop Science 8 (2014): 1168-1174.
- Cowieson AJ., et al. “Phytic acid and phytase: implications for protein utilization by poultry”. Poultry Science 85 (2006): 878-885.
- Liu N., et al. “Effect of diet containing phytate and phytase on the activity and messenger ribonucleic acid expression of carbohydrase and transporter in chickens”. Journal of Animal Science 86 (2008): 3432-3439.
- Woyengo TA., et al. “Nutrient digestibility and performance responses of growing pigs fed phytase-and xylanase-supplemented wheat-based diets”. Journal of Animal Science 86 (2008): 848-857.
- Leytem AB., et al. “Phytate utilization and phosphorus excretion by broiler chickens fed diets containing cereal grains varying in phytate and phytase content”. Animal Feed Science and Technology 146 (2008): 160-168.
- Glover NM. “The Genetic Basis of Phytate, Oligosaccharide Content, and Emergence in Soybean” (Doctoral dissertation, Virginia Tech). Biology (2011).
- Raboy V. “Seed phosphorus and the development of low-phytate crops”. In: Inositol Phosphates: Linking Agriculture and the Environment CABI (2007): 111-132.
- Stevenson-Paulik J., et al. “Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases”. Proceedings of the National Academy of Sciences 102 (2005): 12612-12617.
- Nunes AC., et al. “RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content”. Planta 224 (2006): 125-132.
- Punjabi M., et al. “Development and evaluation of low phytic acid soybean by siRNA triggered seed specific silencing of inositol polyphosphate 6-/3-/5-kinase gene”. Frontiers in Plant Science 9 (2018): 804.
- Curtin SJ., et al. “Genome engineering of crops with designer nucleases”. The Plant Genome 5 (2012): 42-50.
- Bolotin A., et al. “Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin”. Microbiology 151 (2005): 2551-2561.
- Barrangou R., et al. “CRISPR provides acquired resistance against viruses in prokaryotes”. Science 315 (2007): 1709-1712.
- Nishimasu H., et al. “Crystal structure of Cas9 in complex with guide RNA and target DNA”. Cell 156 (2014): 935-949.
- Jiang F., et al. “Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage”. Science 351 (2016): 867-871.
- Shen H., et al. “CRISPR/Cas9-induced double-strand break repair in Arabidopsis nonhomologous end-joining mutants”. G3: Genes, Genomes, Genetics 7 (2017): 193-202.
- Jinek M., et al. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”. Science 337 (2012): 816-821.
- Krishnan V., et al. “AGRODATE’: a rapid Agrobacterium-mediated transient expression tool for gene function analysis in leaf discs”. Journal of Plant Biochemistry and Biotechnology 29 (2020): 294-304.
- Kumari S., et al. “A rapid method for optimization of Agrobacterium-mediated transformation of Indian soybean genotypes”. Indian Journal of Biochemistry and Biophysics 53 (2016): 218-226.
- Michno JM., et al. “CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme”. GM Crops and Food 6 (2015): 243-252.
- Moreno-Mateos MA., et al. “CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo”. Nature Methods 12 (2015): 982-988.
- Liu X., et al. “Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system”. Scientific Reports 6 (2016): 1-9.
- Jinek M., et al. “Structures of Cas9 endonucleases reveal RNA-mediated conformational activation”. Science 343 (2014): 1247997.
- Xia X and Holcik M. “Strong eukaryotic IRESs have weak secondary structure”. PLoS One 4 (2019): e4136.
- Ren X., et al. “Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila”. Cell Reports 9 (2014): 1151-1162.
- Hada A., et al. “Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335”. Plant Biotechnology 33 (2016): 341-350.