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Low phytate soybean: next generation metabolic engineering using CRISPR-Cas 9 genome editing technology

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Abstract

Next-generation metabolic engineering widens the possibility of using plants as biofactories for the mass production of metabolites. CRISPR/Cas9 system is the newest and the most widely adapted genome editing tool for metabolic engineering towards crop improvement. In this RNA guided approach, all sgRNAs are not equally efficient and it is essential to minimize the risk of using ineffective sgRNA producing undesired mutants. In this study, we designed two sgRNAs targeting the terminal step of phytate biosynthesis, GmIPK1- exon 6 (KS1) and 1 (KS2) and validated the efficiency using various in silico tools. We validated the functional efficiency of sgRNAs using AGRODATE (Agrobacterium mediated disc assay for transient expression) prior developing stable mutants. Combined statistical analysis of mutation rate in transient mutants revealed deletions i.e. KS1_sgRNA1 (76.4%) ranging from 1 to 7 nucleotides and insertions accounted for (23.4%) and in case of KS2_sgRNA2, 85.2% deletions were observed which ranged from 1 to 6 nucleotides, while insertions accounted for (13.2%). Sequence analysis of the amplified products revealed the presence of mutations in 12 of the 16 positive transgenic lines (75%). Phytate analysis of sgRNA1 knock-out mutants showed about a 6.6-fold reduction, while sgRNA2 knock-out mutant showed a 7.05-fold reduction in T0 stable soybean knock-out mutants. The strategy undertaken in this research is the first report of a highly effective CRISPR/Cas9 modification system using chimeric gRNA in DS9712 soybean cv., reinforces the importance of developing low phytate soybean with immense potential for food and feed industry.

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Abbreviations

AGRODATE:

Agrobacterium-Mediated disc assay for transient expression

BAP:

6-Benzylaminopurine

CRISPR/Cas9:

Clustered regularly interspaced short palindromic repeats

DSB:

Double strand breaks

EtBr:

Ethidium bromide

sgRNAs:

Single guide RNAs

HR:

Hypersensitive reaction

IBA:

Indole butyric acid

InDels:

Insertion or deletions

IPK1:

Inositol pentakisphosphate 2-kinase 1

LOF:

Loss of function

MFE:

Minimum free energy

MIPS:

myo-Inositol-3-phosphate synthase

NHEJ:

Non-homologous end joining

PA:

Phytic acid

PAM:

Proto-spacer adjacent motifs

PDA:

Photo diode array

TALENS:

Transcription activator-like endonucleases

ZFNs:

Zinc-finger nucleases

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Acknowledgements

We would like to thank National Science Fund (NASF)–ICAR (RNAi/2001) for the grant and Grammarly (https://www.grammarly.com/) for the English language review.

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Authors and Affiliations

  1. Division of Biochemistry, ICAR-Indian Agricultural Research Institute (IARI), New Delhi, 110012, India

    Veda Krishnan, Monica Jolly, Vinutha T. & Archana Sachdev

  2. Department of Biotechnology, Bharathidasan University, Thiruchirappalli, 620024, India

    Veda Krishnan & Manickavasagam M.

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  1. Veda Krishnan
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  2. Monica Jolly
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  4. Manickavasagam M.
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Contributions

VK and AS conceived the idea. VK, M and AS designed the experiments. MJ performed transient transformation and VK performed stable transformation experiments. V assisted in bibliographic survey and HPLC analysis. AS provided critical feedback and shaped the manuscript. All authors discussed the results and contributed to the final manuscript.

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Correspondence to Veda Krishnan or Archana Sachdev.

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Krishnan, V., Jolly, M., T., V. et al. Low phytate soybean: next generation metabolic engineering using CRISPR-Cas 9 genome editing technology. J. Plant Biochem. Biotechnol. 32, 846–861 (2023). https://doi.org/10.1007/s13562-023-00845-1

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