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 Praveen

Department of Biochemistry, Indian Agricultural Research Institute, New Delhi, India

*Corresponding Author: Archana Sachdev, Department of Biochemistry, Indian Agricultural Research Institute, New Delhi, India.

Received: January 27, 2022; Published: February 21, 2022

Abstract

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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Citation

Citation: Archana Sachdev., et al. "Efficient Designing, Validation, and Transformation of GmIPK2 Specific CRISPR/Cas9 Construct for Low-Phytate Soybean". Acta Scientific Agriculture 6.3 (2022): 24-32.

Copyright

Copyright: © 2022 Joshna Jose., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.