Skip to main content
SpringerLink
Log in

Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Main conclusion

Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed.

Abstract

The notorious  cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (βC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (βC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited βC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

Graphical abstract

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

Similar content being viewed by others

Data availability

The sequences of CLCuV were retrieved from the nucleotide domain of NCBI website (https://www.ncbi.nlm.nih.gov/nucleotide/) with accession numbers of KY992859.1, MK370435.1, HE601939.1. The study does not involve the isolation of any new gene. Instead, it involves CRISPR_Cas-mediated knockdown of viral genes into the local cotton variety.

References

  • Ali A, Ahmed Z, Guo Z (2021) 4 cotton growing in Pakistan. Pest Management in Cotton: A Global Perspective 53

  • Asif M, Muhammad R, Akbar W, Toufique M (2017) Response of various cotton genotypes against sucking and bollworm complexes. Pakistan J Agric Agric Eng Vet Sci 33(1):37–45

    Google Scholar 

  • Azam S, Rao AQ, Shad M, Ahmed M, Gul A, Latif A, Ali MA, Husnain T, Shahid AA (2021) Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes. Plant Cell Rep 40(4):707–721

    Article  PubMed  Google Scholar 

  • Bakhsh A, Rao AQ, Shahid AA, Husnain T, Riazuddin S (2009) Insect resistance and risk assessment studies in advance lines of Bt cotton harboring Cry1Ac and Cry2A genes. Am Eurasian J Agric Environ Sci 6(1):1–11

    CAS  Google Scholar 

  • Bakhsh A, Anayol E, Özcan SF, Hussain T, Aasim M, Khawar KM, Özcan S (2015) An insight into cotton genetic engineering (Gossypium hirsutum L.): current endeavors and prospects. Acta Physiol Plant 37(8):1–17

    Article  CAS  Google Scholar 

  • Blecher-Gonen R, Barnett-Itzhaki Z, Jaitin D, Amann-Zalcenstein D, Lara-Astiaso D, Amit I (2013) High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states. Nat Protoc 8(3):539–554

    Article  PubMed  Google Scholar 

  • Cerutti H, Ma X, Msanne J, Repas T (2011) RNA-mediated silencing in algae: biological roles and tools for analysis of gene function. Eukaryot Cell 10(9):1164–1172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cramer P (2021) AlphaFold2 and the future of structural biology. Nat Struct Mol Biol 28(9):704–705

    Article  CAS  PubMed  Google Scholar 

  • DeLano WL (2002) Pymol: An open-source molecular graphics tool. CCP4 Newsl Protein Crystallogr 40(1):82–92

    Google Scholar 

  • Gajula MP, Kumar A, Ijaq J (2016) Protocol for molecular dynamics simulations of proteins. Bio-Protoc 6(23):e2051–e2051

    Google Scholar 

  • Gao W, Long L, Tian X, Xu F, Liu J, Singh PK, Botella JR, Song C (2017) Genome editing in cotton with the CRISPR/Cas9 system. Front Plant Sci 8:1364

    Article  PubMed  PubMed Central  Google Scholar 

  • Gul A, Hussain G, Iqbal A, Rao AQ, Yasmeen A, Shahid N, Ahad A, Latif A, Azam S, Samiullah TR (2020) Constitutive expression of Asparaginase in Gossypium hirsutum triggers insecticidal activity against Bemisia tabaci. Sci Rep 10(1):1–11

    Article  Google Scholar 

  • Hasley JAR, Navet N, Tian M (2021) CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance. PLoS One 16(6):e0253245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hong JK, Suh EJ, Park SR, Park J, Lee Y-H (2021) Multiplex CRISPR/Cas9 mutagenesis of BrVRN1 delays flowering time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Agriculture 11(12):1286

    Article  CAS  Google Scholar 

  • Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu D, Yu Y, Wang C, Long Y, Liu Y, Feng L, Lu D, Liu B, Jia J, Xia R (2021) Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions. Plant Cell 33(9):2950–2964

    Article  PubMed  PubMed Central  Google Scholar 

  • Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596(7873):583–589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kataria SK, Pal RK, Kumar V, Singh P (2019) Population dynamics of whitefly, Bemisia tabaci (Gennadius), as influenced by weather conditions infesting Bt cotton hybrid. J Agrometeorol 21(4):504–509

    Article  Google Scholar 

  • Karplus M, Kuriyan J (2005) Molecular dynamics and protein function. Proc Nat Acad Sci 102(19):6679–6685

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khan ZA, Abdin MZ, Khan JA (2015) Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus. PLoS One 10(3):e0121656

    Article  PubMed  PubMed Central  Google Scholar 

  • Khan AA, El-Sayed A, Akbar A, Mangravita-Novo A, Bibi S, Afzal Z, Norman DJ, Ali GS (2017) A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants. Plant Methods 13(1):1–13

    Article  CAS  Google Scholar 

  • Kheirodin A, Simmons AM, Legaspi JC, Grabarczyk EE, Toews MD, Roberts PM, Chong J-H, Snyder WE, Schmidt JM (2020) Can generalist predators control Bemisia tabaci? InSects 11(11):823

    Article  PubMed  PubMed Central  Google Scholar 

  • Kumar V, AlMomin S, Rahman MH, Shajan A (2020) Use of CRISPR in climate smart/resilient agriculture. In: CRISPR/Cas genome editing. Springer, pp 131–164

  • Kutnjak D, Tamisier L, Adams I, Boonham N, Candresse T, Chiumenti M, De Jonghe K, Kreuze JF, Lefebvre M, Silva G (2021) A primer on the analysis of high-throughput sequencing data for detection of plant viruses. Microorganisms 9(4):841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee JH, Mazarei M, Pfotenhauer AC, Dorrough AB, Poindexter MR, Hewezi T, Lenaghan SC, Graham DE, Stewart CN Jr (2020) Epigenetic footprints of CRISPR/Cas9-mediated genome editing in plants. Front Plant Sci 10:1720

    Article  PubMed  PubMed Central  Google Scholar 

  • Lei Y, Lu L, Liu H-Y, Li S, Xing F, Chen L-L (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7(9):1494–1496

    Article  CAS  PubMed  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 

  • Mahmood TB, Hossan MI, Mahmud S, Shimu MSS, Alam MJ, Bhuyan MMR, Emran TB (2022) Missense mutations in spike protein of SARS‐CoV‐2 delta variant contribute to the alteration in viral structure and interaction with hACE2 receptor. Immun Inflamm Dis 10(9):e683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Majid M, Andleeb S (2019) Designing a multi-epitopic vaccine against the enterotoxigenic Bacteroides fragilis based on immunoinformatics approach. Sci Rep 9(1):1–15

    Article  Google Scholar 

  • Matsuo K, Atsumi G (2019) CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins. Planta 250(2):463–473

    Article  CAS  PubMed  Google Scholar 

  • Misaka BC, Wosula EN, Marchelo-d’Ragga PW, Hvoslef-Eide T, Legg JP (2020) Genetic diversity of Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) colonizing sweet potato and cassava in South Sudan. InSects 11(1):58

    Article  PubMed  PubMed Central  Google Scholar 

  • Mubarik MS, Wang X, Khan SH, Ahmad A, Khan Z, Amjid MW, Razzaq MK, Ali Z, Azhar MT (2021) Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants. GM Crops Food 12(2):647–658

    Article  PubMed  PubMed Central  Google Scholar 

  • Mubin M, Hussain M, Briddon RW, Mansoor S (2011) Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex. Virol J 8(1):1–8

    Article  Google Scholar 

  • Nekrasov V, Wang C, Win J, Lanz C, Weigel D, Kamoun S (2017) Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep 7(1):1–6

    Article  CAS  Google Scholar 

  • Paredes-Montero JR, Hameed U, Zia-Ur-Rehman M, Rasool G, Haider MS, Herrmann H-W, Brown JK (2019) Demographic expansion of the predominant Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) mitotypes associated with the Cotton leaf curl virus epidemic in Pakistan. Ann Entomol Soc Am 112(3):265–280

    Article  Google Scholar 

  • Paredes-Montero JR, Haq Q, Mohamed AA, Brown JK (2021) Phylogeographic and SNPs analyses of Bemisia tabaci B mitotype populations reveal only two of eight haplotypes are invasive. Biology 10(10):1048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reece-Hoyes JS, Walhout AJ (2018) Gateway recombinational cloning. Cold Spring Harb Protoc 2018(1):pdb. top094912

    Article  PubMed  PubMed Central  Google Scholar 

  • Salisu IB, Shahid AA, Yaqoob A, Gul A, Rao AQ (2021) Evaluation the effect of subchronic feeding of transgenic cotton line (CKC1) on the faecal microbiota of albino rabbits. J Anim Physiol Anim Nutr 105(2):354–363

    Article  CAS  Google Scholar 

  • Schachtsiek J, Stehle F (2019) Nicotine-free, nontransgenic tobacco (Nicotiana tabacum L.) edited by CRISPR-Cas9. Plant Biotechnol J 17(12):2228

    Article  PubMed  PubMed Central  Google Scholar 

  • Shad M, Yasmeen A, Azam S, Bakhsh A, Latif A, Shahid N, Sadaqat S, Rao AQ, Shahid AA (2021) Enhancing the resilience of transgenic cotton for insect resistance. Mol Biol Reports 49(6):5315–5323

    Article  Google Scholar 

  • Shah H, Khan K, Khan N, Badshah Y, Ashraf NM, Shabbir M (2022) Impact of deleterious missense PRKCI variants on structural and functional dynamics of protein. Sci Rep 12(1):1–17

    Article  Google Scholar 

  • Sher Z, Majid MU, Hassan S, Batool F, Aftab B, Rashid B (2021) Identification, isolation and characterization of GaCyPI gene in Gossypium arboreum under cotton leaf curl virus disease stress. Phyton 90(6):1613

    Article  Google Scholar 

  • Shukla N, Verma S, Babu GS, Saxena S (2017) Strategy for generic resistance against begomoviruses through RNAi. In: Begomoviruses: occurrence and management in Asia and Africa. Springer, pp 137–155

  • Tennakoon C, Purbojati RW, Sung W-K (2012) BatMis: a fast algorithm for k-mismatch mapping. Bioinformatics 28(16):2122–2128

    Article  CAS  PubMed  Google Scholar 

  • Uniyal AP, Yadav SK, Kumar V (2019) The CRISPR–Cas9, genome editing approach: a promising tool for drafting defense strategy against begomoviruses including cotton leaf curl viruses. J Plant Biochem Biotechnol 28(2):121–132

    Article  CAS  Google Scholar 

  • Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, Darian E, Guvench O, Lopes P, Vorobyov I (2010) CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J Comput Chem 31(4):671–690

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci 112(11):3570–3575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu D, Zhang Y (2011) Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization. Biophys J 101(10):2525–2534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are thankful to KIT Germany for provision of necessary training to execute this study in Pakistan and Humboldt foundation for sponsoring the post doc training of the corresponding author and provision of equipment subsidy for smooth execution of experimentation at CEMB Pakistan.

Funding

The research was conducted by in house facilities of CEMB Center of Excellence in Molecular Biology (CEMB), without any grant. However, the student bench fee was provided by Education Commission (HEC) of Pakistan.

Author information

Author notes

  1. Sana Shakoor and Abdul Qayyum Rao contributed equally to this work.

Authors and Affiliations

  1. Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Bank Road, Lahore, 53700, Pakistan

    Sana Shakoor, Abdul Qayyum Rao, Sara Ajmal, Aneela Yasmeen, Sahar Sadaqat & Tayyab Husnain

  2. Department of Biochemistry and Biotechnology, University of Gujrat, Gujrat, Pakistan

    Muhammad Azmat Ullah Khan

  3. School of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan

    Naeem Mahmood Ashraf

  4. Pacific Biosciences, Bonn, Nordrhein-Westfalen, Deutschland

    Felix Wolter

  5. CureVac Manufacturing GmbH, Tübingen, Baden-Württemberg, Deutschland

    Michael Pacher

Authors
  1. Sana Shakoor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abdul Qayyum Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sara Ajmal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aneela Yasmeen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Muhammad Azmat Ullah Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sahar Sadaqat
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Naeem Mahmood Ashraf
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Felix Wolter
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael Pacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tayyab Husnain
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SS performed all the experiments related to the vector confirmation, genetic transformation, compiled data and wrote the initial draft of manuscript. AQR designed the study, constructed recombinant plasmids, provided technical support and supervised the study. SA, AY and MAUK helped in genetic transformation experiments and recorded the data. SS helped in mutant screening and phenotypic analysis. NMA helped in Bioinformatics analysis, FW assisted in designing of protospacer and strategy, MP helped in the provision of chemicals, TH supervised the overall study, read the manuscript critically and presented it in its current form.

Corresponding author

Correspondence to Abdul Qayyum Rao.

Ethics declarations

Conflict of interest

The authors Sana Shakoor, Abdul Qayyum Rao, Sara Ajmal, Aneela Yasmeen, Muhammad Azmat Ullah Khan, Sahar Sadaqat, Naeem Mahmood Ashraf, Felix Wolter, Michael Pacher and Tayyab Husnain declare that they have no conflicts of interest in submitting or publishing this manuscript entitled “Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants” in the Journal of Planta.

Additional information

Communicated by Anastasios Melis.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 5897 KB)

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

Shakoor, S., Rao, A.Q., Ajmal, S. et al. Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants. Planta 258, 79 (2023). https://doi.org/10.1007/s00425-023-04233-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00425-023-04233-w

Keywords

Search

Navigation