Efficient Designing, Validation, and Transformation of GmIPK2 Specific CRISPR/Cas9 Construct for Low-Phytate Soybean
Archana Sachdev, Joshna Jose*, Monica Jolly, Veda Krishnan,
Urvi Mehrotra, Smrutirekha Sahu, Vinutha T, Anil Dahuja, Shelly
Department of Biochemistry, Indian Agricultural Research Institute, New Delhi, India
*Corresponding Author: Archana Sachdev, Department of Biochemistry, 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
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