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CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut

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Abstract

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

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References

  • Bánfalvi Z, Csákvári E, Villányi V, Kondrák M (2020) Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation. BMC Biotechnol 20:1–10

    Article  Google Scholar 

  • Bao A, Chen H, Chen L, Chen S, Hao Q, Guo W, Qiu D, Shan Z, Yang Z, Yuan S (2019) CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean. BMC Plant Biol 19:1–12

    Article  CAS  Google Scholar 

  • Bertioli DJ, Jenkins J, Clevenger J, Dudchenko O, Gao D, Seijo G, Leal-Bertioli SC, Ren L, Farmer AD, Pandey MK (2019) The genome sequence of segmental allotetraploid peanut Arachis hypogaea. Nat Genet 51:877–884

    Article  CAS  PubMed  Google Scholar 

  • Biswas S, Wahl NJ, Thomson MJ, Cason JM, McCutchen BF, Septiningsih EM (2022) Optimization of protoplast isolation and transformation for a pilot study of genome editing in peanut by targeting the allergen gene Ara h 2. Int J Mol Sci 23:837

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Biswas S, Ibarra O, Shaphek M, Molina-Risco M, Faion‐Molina M, Bellinatti‐Della Gracia M, Thomson MJ, Septiningsih EM (2023) Increasing the level of resistant starch in ‘Presidio’rice through multiplex CRISPR–Cas9 gene editing of starch branching enzyme genes. Plant Genome 16:e20225

    Article  CAS  PubMed  Google Scholar 

  • Cai Y, Chen L, Zhang Y, Yuan S, Su Q, Sun S, Wu C, Yao W, Han T, Hou W (2020) Target base editing in soybean using a modified CRISPR/Cas9 system. Plant Biotechnol. J. 18:1996

  • Castel B, Tomlinson L, Locci F, Yang Y, Jones JD (2019) Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis. PLoS ONE 14:e0204778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Che P, Chang S, Simon MK, Zhang Z, Shaharyar A, Ourada J, O’Neill D, Torres-Mendoza M, Guo Y, Marasigan KM (2021) Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants. Plant J 106:817–830

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Conant D, Hsiau T, Rossi N, Oki J, Maures T, Waite K, Yang J, Joshi S, Kelso R, Holden K (2022) Inference of CRISPR edits from Sanger trace data. CRISPR J 5:123–130

    Article  CAS  PubMed  Google Scholar 

  • Dayal S, Lavanya M, Devi P, Sharma K (2003) An efficient protocol for shoot regeneration and genetic transformation of pigeonpea [Cajanus cajan (L.) Millsp.] Using leaf explants. Plant Cell Rep 21:1072–1079

    Article  CAS  PubMed  Google Scholar 

  • Gao R, Feyissa BA, Croft M, Hannoufa A (2018) Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa. Planta 247:1043–1050

    Article  CAS  PubMed  Google Scholar 

  • Ghosh G, Ganguly S, Purohit A, Chaudhuri RK, Das S, Chakraborti D (2017) Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera. Plant Cell Rep 36:1037–1051

    Article  CAS  PubMed  Google Scholar 

  • Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V (2020) A modular cloning toolkit for genome editing in plants. BMC Plant Biol 20:1–10

    Article  Google Scholar 

  • Hickey LT, A NH, Robinson H, Jackson SA, Leal-Bertioli SCM, Tester M, Gao C, Godwin ID, Hayes BJ, Wulff BBH (2019) Breeding crops to feed 10 billion. Nat Biotechnol 37:744–754

    Article  CAS  PubMed  Google Scholar 

  • Hooghvorst I, López-Cristoffanini C, Nogués S (2019) Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon. Sci Rep 9:17077

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  • Janssens C, Havlik P, Krisztin T, Baker J, Frank S, Hasegawa T, Leclere D, Ohrel S, Ragnauth S, Schmid E, Valin H, Van Lipzig N, Maertens M (2020) Global hunger and climate change adaptation through international trade.Nat. Clim Change 10:829–835

    ADS  Google Scholar 

  • Ji J, Zhang C, Sun Z, Wang L, Duanmu D, Fan Q (2019) Genome editing in cowpea Vigna unguiculata using CRISPR-Cas9. Int J Mol Sci 20:2471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang W, Yang B, Weeks DP (2014) Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations. PLoS ONE 9:e99225

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  • Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  • Kamens J (2015) The Addgene repository: an international nonprofit plasmid and data resource. Nucleic Acids Res 43:D1152–D1157

    Article  CAS  PubMed  Google Scholar 

  • Karmakar S, Molla KA, Gayen D, Karmakar A, Das K, Sarkar SN, Datta K, Datta SK (2019) Development of a rapid and highly efficient Agrobacterium-mediated transformation system for pigeon pea [Cajanus cajan (L.) Millsp]. GM Crops Food 10:115–138

    Article  PubMed  PubMed Central  Google Scholar 

  • Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinform 28:1647–1649

    Article  Google Scholar 

  • Kulshreshtha SN, Wheaton EE (2018) Sustainable Agriculture and Climate ChangeMDPI Basel

  • Kumar S, Rymarquis LA, Ezura H, Nekrasov V (2021) CRISPR-Cas in agriculture: opportunities and challenges. Front. Plant Sci 12:672329

    Google Scholar 

  • Labun K, Montague TG, Krause M, Torres Cleuren YN, Tjeldnes H, Valen E (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47:W171–W174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Y, Chen Z, Zhang C, Guo J, Liu Q, Yin Y, Hu Y, Xia H, Li B, Sun X (2024) Gene editing of ZmGA20ox3 improves plant architecture and drought tolerance in maize. Plant Cell Rep 43:18

    Article  CAS  Google Scholar 

  • Lowder LG, Zhang D, Baltes NJ, Paul JW III, Tang X, Zheng X, Voytas DF, Hsieh T-F, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169:971–985

    Article  PubMed  PubMed Central  Google Scholar 

  • Lu QSM, Tian L (2022) An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes. BMC Biotechnol 22:7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284

    Article  CAS  PubMed  Google Scholar 

  • Mainkar P, Manape TK, Satheesh V, Anandhan S (2023) CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.). Front. Plant Sci. 14

  • Malhotra N (2024) Application of genome editing in pulses. Applications of Genome Engineering in Plants, pp 326–338

  • Mehta R, Radhakrishnan T, Kumar A, Yadav R, Dobaria JR, Thirumalaisamy PP, Jain RK, Chigurupati P (2013) Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation. Indian J Virol 24:205–213

    Article  PubMed  PubMed Central  Google Scholar 

  • Meng Y, Hou Y, Wang H, Ji R, Liu B, Wen J, Niu L, Lin H (2017) Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula. Plant Cell Rep 36:371–374

    Article  CAS  PubMed  Google Scholar 

  • Mikami M, Toki S, Endo M (2016) Precision targeted mutagenesis via Cas9 paired nickases in rice. Plant Cell Physiol 57:1058–1068

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Móring A, Hooda S, Raghuram N, Adhya TK, Ahmad A, Bandyopadhyay SK, Barsby T, Beig G, Bentley AR, Bhatia A (2021) Nitrogen challenges and opportunities for agricultural and environmental science in India. Front Sustain food syst 5:505347

    Article  Google Scholar 

  • Naim F, Dugdale B, Kleidon J, Brinin A, Shand K, Waterhouse P, Dale J (2018) Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9. Transgenic Res 27:451–460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Neelakandan AK, Wright DA, Traore SM, Chen X, Spalding MH, He G (2022a) CRISPR/Cas9 based site-specific modification of FAD2 cis-regulatory motifs in peanut (Arachis hypogaea L). Front Genet 13:849961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Neelakandan AK, Subedi B, Traore SM, Binagwa P, Wright DA, He G (2022b) Base editing in peanut using CRISPR/nCas9. Front. genome ed. 4:901444

  • Ntui VO, Tripathi JN, Tripathi L (2020) Robust CRISPR/Cas9 mediated genome editing tool for banana and plantain (Musa spp.). Curr. Plant Biol 21:100128

    Google Scholar 

  • Ochatt S, Conreux C, Moussa Mcolo R, Despierre G, Magnin-Robert J-B, Raffiot B (2018) Phytosulfokine-alpha, an enhancer of in vitro regeneration competence in recalcitrant legumes. Plant Cell, Tissue Organ Cult. 135:189–201

  • Odipio J, Alicai T, Ingelbrecht I, Nusinow DA, Bart R, Taylor NJ (2017) Efficient CRISPR/Cas9 genome editing of phytoene desaturase in cassava. Front Plant Sci 8:1780

    Article  PubMed  PubMed Central  Google Scholar 

  • Ojiewo CO, Janila P, Bhatnagar-Mathur P, Pandey MK, Desmae H, Okori P, Mwololo J, Ajeigbe H, Njuguna-Mungai E, Muricho G (2020) Advances in crop improvement and delivery research for nutritional quality and health benefits of groundnut (Arachis hypogaea L.). Front. Plant Sci 11:29

    Google Scholar 

  • Poczai P, Varga I, Laos M, Cseh A, Bell N, Valkonen JP, Hyvönen J (2013) Advances in plant gene-targeted and functional markers: a review. Plant Methods 9:1–32

    Article  Google Scholar 

  • Prasad K, Bhatnagar-Mathur P, Waliyar F, Sharma KK (2013) Overexpression of a chitinase gene in transgenic peanut confers enhanced resistance to major soil borne and foliar fungal pathogens. J Plant Biochem Biotechnol 22:222–233

    Article  CAS  Google Scholar 

  • Pratap A, Prajapati U, Singh CM, Gupta S, Rathore M, Malviya N, Tomar R, Gupta AK, Tripathi S, Singh NP (2018) Potential, constraints and applications of in vitro methods in improving grain legumes. Plant Breed 137:235–249

    Article  Google Scholar 

  • Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17:471–482

    Article  CAS  PubMed  Google Scholar 

  • Rajyaguru R, Maheshala N, Gangadhara K (2024) Genetic improvement in peanut: role of Genetic Engineering. Genetic Engineering of Crop Plants for Food and Health Security, vol 1. Springer Nature Singapore, Singapore, pp 271–288

    Google Scholar 

  • Sharma KK, Bhatnagar-Mathur P (2006) Peanut (Arachis hypogaea l). Agrobacterium Protocols :347–358

  • Sharma K, Sreelatha G, Dayal S (2006) Pigeonpea (Cajanus cajan L. Millsp). Agrobacterium Protocols 2006:359–368

    Article  Google Scholar 

  • Shu H, Luo Z, Peng Z, Wang J (2020) The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation. BMC Plant Biol 20:1–15

    Article  Google Scholar 

  • Singh N, Jain P, Ujinwal M, Langyan S (2022) Escalate protein plates from legumes for sustainable human nutrition. Front Nutr 9:977986

    Article  PubMed  PubMed Central  Google Scholar 

  • Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, Xi Y (2015) Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep 5:10342

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  • Tian L (2015) Recent advances in understanding carotenoid-derived signaling molecules in regulating plant growth and development. Front. Plant Sci 6:790

    Google Scholar 

  • Varshney RK, Chen W, Li Y, Bharti AK, Saxena RK, Schlueter JA, Donoghue MT, Azam S, Fan G, Whaley AM (2012) Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers. Nat Biotechnol 30:83

    Article  CAS  Google Scholar 

  • Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y-G, Zhao K (2016) Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS ONE 11:e0154027

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang W, Pan Q, Tian B, He F, Chen Y, Bai G, Akhunova A, Trick HN, Akhunov E (2019a) Gene editing of the wheat homologs of TONNEAU 1-recruiting motif encoding gene affects grain shape and weight in wheat. Plant J 100:251–264

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang L, Rubio MC, Xin X, Zhang B, Fan Q, Wang Q, Ning G, Becana M, Duanmu D (2019b) CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation. New Phytol 224:818–832

    Article  CAS  PubMed  Google Scholar 

  • Wolabu TW, Cong L, Park J-J, Bao Q, Chen M, Sun J, Xu B, Ge Y, Chai M, Liu Z (2020) Development of a highly efficient multiplex genome editing system in outcrossing tetraploid alfalfa (Medicago sativa). Front Plant Sci 11:1063

    Article  PubMed  PubMed Central  Google Scholar 

  • Yuan M, Zhu J, Gong L, He L, Lee C, Han S, Chen C, He G (2019) Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing. BMC Biotechnol 19:1–7

    Article  Google Scholar 

  • Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N (2014) The CRISPR/C as9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807

    Article  CAS  PubMed  Google Scholar 

  • Zhang S, Zhang R, Gao J, Song G, Li J, Li W, Qi Y, Li Y, Li G (2021) CRISPR/Cas9-mediated genome editing for wheat grain quality improvement. Plant Biotechnol J 19:1684

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhuang W, Chen H, Yang M, Wang J, Pandey MK, Zhang C, Chang W-C, Zhang L, Zhang X, Tang R (2019) The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication. Nat Genet 51:865–876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was carried out with the aid of a grant from the Start-up Research Grant (SRG) (File No. SRG/2021/000422) from the Science and Engineering Research Board (SERB), Govt. of India to KY. The authors express their gratitude to Dr Prakash Gangashetty, the Pigeonpea breeder at ICRISAT, and Dr Janila Pasupuleti, the Groundnut breeder at ICRISAT, for generously supplying the pigeonpea and groundnut seeds required for plant transformation.

Funding

This work was carried out with the aid of a grant from the Start-up Research Grant (SRG) (File No. SRG/2021/000422) from the Science and Engineering Research Board (SERB), Govt. of India.

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  1. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502324, India

    Kalyani Prasad, Harika Gadeela, Pradeep Reddy Bommineni, Palakolanu Sudhakar Reddy, Wricha Tyagi & Kalenahalli Yogendra

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  1. Kalyani Prasad
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Contributions

Conceptualization: KY and PSR; methodology: KP, HG, PRB, and KY; software: KY and PSR; data analysis: KY, and WT; writing—original draft preparation: KP, HG, and KY; writing—review and editing: KY, WT, and PSR; supervision and funding acquisition: KY. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Kalenahalli Yogendra.

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Prasad, K., Gadeela, H., Bommineni, P.R. et al. CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut. Funct Integr Genomics 24, 57 (2024). https://doi.org/10.1007/s10142-024-01336-9

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