Skip to main content
SpringerLink
Log in

In silico analysis and designing gRNA constructs for the precise modification of the OsTMS5 gene in rice (Oryza sativa L.): a comprehensive study and construct development for crop improvement

  • Original Paper
  • Published:
Cereal Research Communications Aims and scope Submit manuscript

Abstract

The CRISPR/Cas9 system represents a state-of-the-art technology for precise genome editing in plants. In this study, we performed in silico and evolutionary analyses, as well as designed guide RNA (gRNA) constructs for the precise modification of the thermosensitive genic male sterile (OsTMS5) gene using the CRISPR/Cas9 system in rice (Oryza sativa L.). The OsTMS5 promoter harbours a diverse array of cis-elements, which are linked to light responsiveness, hormonal regulation, and stress-related signaling. Further, expression pattern of OsTMS5 revealed that OsTMS5 exhibited responsiveness to hormones and was activated across diverse tissues and developmental stages in rice. In addition, we meticulously designed gRNA with a length of 20 base pairs. This design process was conducted using the CRISPR-P v2.0 online platform. The target of these gRNAs was the rice OsTMS5 gene. The selection of the top two gRNAs was made after conducting a thorough evaluation, which included assessing factors such as on-score value, minimum off-target score, GC content, potential off-target sites, and genomic location. Furthermore, two types of entry vectors were utilized, and the pMDC99 vector served as the destination vector for plant transformation. Following the annealing and ligation of the gRNAs through LR recombination, the resulting plasmid was named as “pMDC99-eSPCas9 + OsU6-OsTMS5-target1-gRNA + OsU6-OsTMS5-target2-gRNA”. Subsequently, this plasmid obtained from the third LR recombination was introduced into Agrobacterium EHA105 for the purpose of conducting rice transformation. Therefore, these constructs have the potential for use not only in molecular genetic analyses and molecular breeding in rice but also in a wide range of other crop species.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

Data availability

Data are available in the manuscript and in the Supplementary Materials.

References

  • Abbas A, Yu P, Sun L, Yang Z, Chen D, Cheng S, Cao L (2021) Exploiting genic male sterility in rice: from molecular dissection to breeding applications. Front Plant Sci 12:629314

    Article  PubMed  PubMed Central  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410

    Article  CAS  PubMed  Google Scholar 

  • Andrade JM, Pobre V, Silva IJ, Domingues S, Arraiano CM (2009) The role of 3′–5′ exoribonucleases in RNA degradation. Prog Mol Biol Transl Sci 85:187–229

    Article  CAS  PubMed  Google Scholar 

  • Anjala K, Augustine R (2022) Designing of guide rna constructs for crispr/cas9-mediated editing of rice transcription factor osmads26 for enhancing drought tolerance. J Appl Biol Biotech 11(1):176–182

    Google Scholar 

  • Ashraf M, Ahmad MSA, Öztürk M, Aksoy A (2012) Crop improvement through different means: challenges and prospects. Crop Prod Agric Improv. https://doi.org/10.1007/978-94-007-4116-4_1

    Article  Google Scholar 

  • Bailey TL, Johnson J, Grant CE, Noble WS (2015) The MEME suite. Nucleic Acids Res 43(W1):W39–W49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barman A, Deb B, Chakraborty S (2020) A glance at genome editing with CRISPR–Cas9 technology. Curr Genet 66:447–462

    Article  CAS  PubMed  Google Scholar 

  • Boettcher M, McManus MT (2015) Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Mol Cell 58(4):575–585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bolotin A, Quinquis B, Sorokin A, Ehrlich SD (2005) Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151(8):2551–2561

    Article  CAS  PubMed  Google Scholar 

  • Budar F, Pelletier G (2001) Male sterility in plants: occurrence, determinism, significance and use. C R De L’acad Des Sci Ser III Sci De La Vie 324(6):543–550

    CAS  Google Scholar 

  • Cao L, Zhan X (2014) Chinese experiences in breeding three-line, two-line and super hybrid rice. In: Yan WG, Bao JS (eds) Rice: germplasm, genetics and improvement. InTech, Rijeka, pp 279–308

    Google Scholar 

  • Chen L, Liu Y-G (2014) Male sterility and fertility restoration in crops. Annu Rev Plant Biol 65:579–606

    Article  CAS  PubMed  Google Scholar 

  • Chen X, Tian Y, Lu X (2018) Breeding of the dormant thermosensitive genic male-sterile lines of early rice to overcome pre-harvest sprouting of the hybrid seeds. Agronomy 8(9):191

    Article  CAS  Google Scholar 

  • Clamp M, Cuff J, Searle SM, Barton GJ (2004) The jalview java alignment editor. Bioinformatics 20(3):426–427

    Article  CAS  PubMed  Google Scholar 

  • Danshina PV, Geyer CB, Dai Q, Goulding EH, Willis WD, Kitto GB, McCarrey JR, Eddy E, O’Brien DA (2010) Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice. Biol Reprod 82(1):136–145

    Article  CAS  PubMed  Google Scholar 

  • Deutscher MP, Li Z (2000) Exoribonucleases and their multiple roles in RNA metabolism

  • Fang Y, Yang J, Guo X, Qin Y, Zhou H, Liao S, Liu F, Qin B, Zhuang C, Li R (2022) CRISPR/Cas9-induced mutagenesis of TMS5 confers thermosensitive genic male sterility by influencing protein expression in rice (Oryza sativa L.). Int J Mol Sci 23(15):8354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Farinati S, Draga S, Betto A, Palumbo F, Vannozzi A, Lucchin M, Barcaccia G (2023) Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications. Front Plant Sci 14:1223861

    Article  PubMed  PubMed Central  Google Scholar 

  • Gonzalez Guzman M, Cellini F, Fotopoulos V, Balestrini R, Arbona V (2022) New approaches to improve crop tolerance to biotic and abiotic stresses. Physiol Plant 174(1):e13547

    Article  CAS  PubMed  Google Scholar 

  • Guo A-Y, Zhu Q-H, Chen X, Luo J-C (2007) GSDS: a gene structure display server. Yi Chuan= Hereditas 29(8):1023–1026

    Article  CAS  PubMed  Google Scholar 

  • Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217:109–119

    Article  PubMed  Google Scholar 

  • Hiei Y, Komari T (2006) Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 85:271–283

    Article  CAS  Google Scholar 

  • Hussain A, Ding X, Alariqi M, Manghwar H, Hui F, Li Y, Cheng J, Wu C, Cao J, Jin S (2021) Herbicide resistance: another hot agronomic trait for plant genome editing. Plants 10(4):621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Janick J (1998) Hybrids in horticultural crops. Concepts Breed Heterosis Crop Plants 25:45–56

    Google Scholar 

  • Jiang F, Doudna JA (2017) CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys 46:505–529

    Article  CAS  PubMed  Google Scholar 

  • Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jin J, Gui S, Li Q, Wang Y, Zhang H, Zhu Z, Chen H, Sun Y, Zou Y, Huang X (2020) The transcription factor GATA10 regulates fertility conversion of a two-line hybrid tms5 mutant rice via the modulation of UbL40 expression. J Integr Plant Biol 62(7):1034–1056

    Article  CAS  PubMed  Google Scholar 

  • Joshi R, Karan R, Singla-Pareek SL, Pareek A (2016) Ectopic expression of Pokkali phosphoglycerate kinase-2 (OsPGK2-P) improves yield in tobacco plants under salinity stress. Plant Cell Rep 35:27–41

    Article  CAS  PubMed  Google Scholar 

  • Kawamura Y, Asai K, Ishii S, Nagano N (2003) Systematic analyses of P-loop containing nucleotide triphosphate hydrolase superfamily based on sequence, structure and function. Genome Infor 14:581–582

    Google Scholar 

  • Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khatodia S, Bhatotia K, Passricha N, Khurana S, Tuteja N (2016) The CRISPR/Cas genome-editing tool: application in improvement of crops. Front Plant Sci 7:506

    Article  PubMed  PubMed Central  Google Scholar 

  • Khush GS (2013) Strategies for increasing the yield potential of cereals: case of rice as an example. Plant Breed 132(5):433–436

    Article  CAS  Google Scholar 

  • Kozlowski LP (2016) IPC—isoelectric point calculator. Biol Direct 11(1):1–16

    Article  Google Scholar 

  • Kumar S, Tamura K, Nei M (1994) MEGA: molecular evolutionary genetics analysis software for microcomputers. Bioinformatics 10(2):189–191

    Article  CAS  Google Scholar 

  • Kumar M, Kesawat MS, Ali A, Lee S-C, Gill SS, Kim HU (2019) Integration of abscisic acid signaling with other signaling pathways in plant stress responses and development. Plants 8(12):592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar D, Yadav A, Ahmad R, Dwivedi UN, Yadav K (2022) CRISPR-based genome editing for nutrient enrichment in crops: a promising approach toward global food security. Front Genet 13:932859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Lawton-Rauh A (2003) Evolutionary dynamics of duplicated genes in plants. Mol Phylogenetics Evol 29(3):396–409

    Article  CAS  Google Scholar 

  • Lei D, Tang W, Xie Z, Liu H, Chen L (2014) Solutions to insecurity problems in seed production of two-line hybrid rice. Agric Sci Technol 15(7):1160

    Google Scholar 

  • Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325–327

    Article  CAS  PubMed  PubMed Central  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(2):971–985

    Article  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(8):1274–1284

    Article  CAS  PubMed  Google Scholar 

  • Metsalu T, Vilo J (2015) ClustVis: a web tool for visualizing clustering of multivariate data using principal component analysis and heatmap. Nucleic Acids Res 43(W1):W566–W570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moore KL (2003) The biology and enzymology of protein tyrosine O-sulfation. J Biol Chem 278(27):24243–24246

    Article  CAS  PubMed  Google Scholar 

  • Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31(7):1120–1123

    Article  CAS  PubMed  Google Scholar 

  • Nejad ES, Askari H, Soltani S (2012) Regulatory TGACG-motif may elicit the secondary metabolite production through inhibition of active Cyclin-dependent kinase/Cyclin complex. Plant Omics 5(6):553–558

    CAS  Google Scholar 

  • Pathak H, Kumar M, Molla KA, Chakraborty K (2021) Abiotic stresses in rice production: impacts and management. Oryza 58(4):103–125

    Article  Google Scholar 

  • Radha B, Sunitha NC, Sah RP, TP MA, Krishna G, Umesh DK, Thomas S, Anilkumar C, Upadhyay S, Kumar A (2023) Physiological and molecular implications of multiple abiotic stresses on yield and quality of rice. Front Plant Sci 13:996514

    Article  PubMed  PubMed Central  Google Scholar 

  • Raghuvanshi S, Kapoor M, Tyagi S, Kapoor S, Khurana P, Khurana J, Tyagi A (2010) Rice genomics moves ahead. Mol Breed 26:257–273

    Article  CAS  Google Scholar 

  • Raman KV, Aggarwal D, Rao SR, Sreevathsa R, Singh A, Abdin M, Mohapatra T, Pattanayak D (2018) Rapid and efficient Agrobacterium mediated transformation of early scutellum derived calli of indica rice

  • Rao MJ, Wang L (2021) CRISPR/Cas9 technology for improving agronomic traits and future prospective in agriculture. Planta 254:1–16

    Article  Google Scholar 

  • Ricroch A, Clairand P, Harwood W (2017) Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture. Emerg Top Life Sci 1(2):169–182

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roy SW, Penny D (2007) Patterns of intron loss and gain in plants: intron loss–dominated evolution and genome-wide comparison of O. sativa and A. thaliana. Mol Biol Evol 24(1):171–181

    Article  CAS  PubMed  Google Scholar 

  • Sawhney VK, Shukla A (1994) Male sterility in flowering plants: Are plant growth substances involved? Am J Bot 81(12):1640–1647

    Article  Google Scholar 

  • Sedeek KE, Mahas A, Mahfouz M (2019) Plant genome engineering for targeted improvement of crop traits. Front Plant Sci 10:114

    Article  PubMed  PubMed Central  Google Scholar 

  • Shalaeva DN, Cherepanov DA, Galperin MY, Golovin AV, Mulkidjanian AY (2018) Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases in relation to the basic catalytic mechanism. Elife 7:e37373

    Article  PubMed  PubMed Central  Google Scholar 

  • Sibéril Y, Doireau P, Gantet P (2001) Plant bZIP G-box binding factors. Modular structure and activation mechanisms. Eur J Biochem 268(22):5655–5666

    Article  PubMed  Google Scholar 

  • Sun S, Wang D, Li J, Lei Y, Li G, Cai W, Zhao X, Liang W, Zhang D (2021) Transcriptome analysis reveals photoperiod-associated genes expressed in rice anthers. Front Plant Sci 12:621561

    Article  PubMed  PubMed Central  Google Scholar 

  • Sun Y, Fu M, Ang Y, Zhu L, Wei L, He Y, Zeng H (2022) Combined analysis of transcriptome and metabolome reveals that sugar, lipid, and phenylpropane metabolism are essential for male fertility in temperature-induced male sterile rice. Front Plant Sci 13:945105

    Article  PubMed  PubMed Central  Google Scholar 

  • Sze H, Palanivelu R, Harper JF, Johnson MA (2021) Holistic insights from pollen omics: co-opting stress-responsive genes and ER-mediated proteostasis for male fertility. Plant Physiol 187(4):2361–2380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tahir T, Ali Q, Rashid M, Malik A (2020) The journey of CRISPR–Cas9 from bacterial defense mechanism to a gene editing tool in both animals and plants. Biol Clin Sci Res J. https://doi.org/10.54112/bcsrj.v2020i1.17

    Article  Google Scholar 

  • Tang H, Song Y, Guo J, Wang J, Zhang L, Niu N, Ma S, Zhang G, Zhao H (2018) Physiological and metabolome changes during anther development in wheat (Triticum aestivum L.). Plant Physiol Biochem 132:18–32

    Article  CAS  PubMed  Google Scholar 

  • Van Eck J (2020) Applying gene editing to tailor precise genetic modifications in plants. J Biol Chem 295(38):13267–13276

    Article  PubMed  PubMed Central  Google Scholar 

  • Waltz E (2018) With a free pass, CRISPR-edited plants reach market in record time. Nat Biotechnol 36(1):6–8

    Article  CAS  PubMed  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(4):e0154027

    Article  PubMed  PubMed Central  Google Scholar 

  • Wen J, Wang L, Wang J, Zeng Y, Xu Y, Li S (2019) The transcription factor OsbHLH138 regulates thermosensitive genic male sterility in rice via activation of TMS5. Theor Appl Genet 132:1721–1732

    Article  CAS  PubMed  Google Scholar 

  • Wen J, Zeng Y, Chen Y, Fan F, Li S (2021) Genic male sterility increases rice drought tolerance. Plant Sci 312:111057

    Article  CAS  PubMed  Google Scholar 

  • William Roy S, Gilbert W (2006) The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genet 7(3):211–221

    Article  CAS  Google Scholar 

  • Xie K, Minkenberg B, Yang Y (2014) Targeted gene mutation in rice using a CRISPR–Cas9 system. Bio Protocol 4(17):e1225–e1225

    Article  Google Scholar 

  • Xu G, Guo C, Shan H, Kong H (2012) Divergence of duplicate genes in exon–intron structure. Proc Natl Acad Sci USA 109(4):1187–1192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu Z-Y, Kim DH, Hwang I (2013) ABA homeostasis and signaling involving multiple subcellular compartments and multiple receptors. Plant Cell Rep 32:807–813

    Article  CAS  PubMed  Google Scholar 

  • Yoshihara T, Washida H, Takaiwa F (1996) A 45-bp proximal region containing AACA and GCN4 motif is sufficient to confer endosperm-specific expression of the rice storage protein glutelin gene, GluA-3. FEBS Lett 383(3):213–218

    Article  CAS  PubMed  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(6):797–807

    Article  CAS  PubMed  Google Scholar 

  • Zhang C, Ren MY, Han WJ, Zhang YF, Huang MJ, Wu SY, Huang J, Wang Y, Zhang Z, Yang ZN (2022) Slow development allows redundant genes to restore the fertility of rpg1, a TGMS line in Arabidopsis. Plant J 109(6):1375–1385

    Article  CAS  PubMed  Google Scholar 

  • Zhou H, Zhou M, Yang Y, Li J, Zhu L, Jiang D, Dong J, Liu Q, Gu L, Zhou L (2014) RNase ZS1 processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice. Nat Commun 5(1):4884

    Article  CAS  PubMed  Google Scholar 

  • Zhou H, He M, Li J, Chen L, Huang Z, Zheng S, Zhu L, Ni E, Jiang D, Zhao B (2016) Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci Rep 6(1):37395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are thankful to the Department of Science and Technology, Government of India, New Delhi, for providing grant for student under INSPIRE fellowship and also greatly acknowledge to ICAR-NRRI Cuttack for providing all the laboratory facilities. The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP2024R347), King Saud University, Riyadh, Saudi Arabia.

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Biotechnology, Orissa University of Agriculture and Technology, Bhubaneswar, 751003, India

    Laxmipreeya Behera & Kailash Ch. Samal

  2. Crop Improvement Division, ICAR-National Rice Research Institute, Cuttack, Odisha, 753006, India

    C. Parmeswaran & Sanghamitra Samantaray

  3. ICAR - National Institute of Biotic Stress Management, Raipur, 493225, India

    Pawan Kumar Agrawal

  4. Crop Improvement Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    V. Mohan Murali Achary

  5. Department of Genetics and Plant Breeding, Orissa University of Agriculture and Technology, Bhubaneswar, 751003, India

    Manasi Dash

  6. Department of Agronomy, Orissa University of Agriculture and Technology, Bhubaneswar, 751003, India

    Sanat Kumar Dwibedi

  7. Department of Plant Physiology, Orissa University of Agriculture and Technology, Bhubaneswar, 751003, India

    Raj Kumari Bhol

  8. Krishi Vigyan Kendra, Bikaner II, Swami Keshwanand Rajasthan Agricultural University, Bikaner, Rajasthan, 334603, India

    Bhagwat Singh Kherawat

  9. Department of Life Science, Dongguk University, 10326, Dong-guIlsan, Republic of South Korea

    Sang-Min Chung

  10. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia

    Manzer H. Siddiqui & Saud Alamri

  11. Department of Genetics and Plant Breeding, Faculty of Agriculture, Sri Sri University, Cuttack, Odisha, 754006, India

    Mahipal Singh Kesawat

Authors
  1. Laxmipreeya Behera
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kailash Ch. Samal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Parmeswaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pawan Kumar Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V. Mohan Murali Achary
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Manasi Dash
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sanat Kumar Dwibedi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Raj Kumari Bhol
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bhagwat Singh Kherawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sang-Min Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Manzer H. Siddiqui
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Saud Alamri
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mahipal Singh Kesawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Sanghamitra Samantaray
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.B., M.S.K., and S.S. helped in conceptualization, formal analysis, designed and wrote the manuscript, and review & editing; S.S. and M.S.K. supervised the study; and K.C.S., P.C., P.K.A., V.M.M.A., M.D., S.K.D., R.K.B., B.S.K., S.-M.C., M.H.S., and S.A provided valuable feedback to this study. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Mahipal Singh Kesawat or Sanghamitra Samantaray.

Ethics declarations

Conflicts of interest

The authors declare no conflict of interest.

Ethical approval

Not applicable.

Informed consent

Not applicable.

Institutional review board

Not applicable.

Additional information

Communicated by Janusz Zimny.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Behera, L., Samal, K.C., Parmeswaran, C. et al. In silico analysis and designing gRNA constructs for the precise modification of the OsTMS5 gene in rice (Oryza sativa L.): a comprehensive study and construct development for crop improvement. CEREAL RESEARCH COMMUNICATIONS (2024). https://doi.org/10.1007/s42976-024-00507-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s42976-024-00507-5

Keywords

Search

Navigation