Plant Protect. Sci., 2020, 56(3):154-162 | DOI: 10.17221/105/2019-PPS

Evaluation of the CRISPR/Cas9 system for the development of resistance against Cotton leaf curl virus in model plantsOriginal Paper

Sehrish Khan*,1, Muhammad Shahid Mahmood1, Sajjad ur Rahman1, Farzana Rizvi2, Aftab Ahmad*,3
1 Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan
2 Department of Pathology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan
3 Department of Biochemistry/U.S.-Pakistan Center for Advanced Studies in Agriculture and Food Security, University of Agriculture Faisalabad, Faisalabad, Pakistan

Over the last decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR/Cas9 system has been used by biologists in various fields. In plant biology, the technology is being utilised to manufacture transgenic plants resistant to different diseases. In Pakistan, the Cotton leaf curl virus (CLCuV) (a begomovirus) affects cotton plants causing significant loss to the economy of this agriculturally based country. In the present study, we use the CRISPR/Cas9 system in Nicotiana benthamiana Domin (a model plant) to develop resistance against CLCuV. An interesting facet of the study was the comparison of two constructs (pHSE401 and pKSE401) with regards to their efficacy in the virus inhibition. The pKSE401 vector contained a Cas9 nuclease and two guide RNAs (gRNAs), one targeting the Replication associated protein (Rep) gene and the other targeted the βC1 gene of the Betasatellite. The vector pHSE401 had only one sgRNA that targeted the (Rep) gene. Both genes that are intended to be targeted play important roles in the replication of CLCuV. Plants infiltrated with pKSE401 exhibited a delay in the development of the symptoms of the disease and showed lower virus titres. Our proposed multiplexing approach gave efficient results of the resistance in the model plants, and the results in this communication may be extended to the CRISPR/Cas9 based editing of cotton plants.

Keywords: Nicotiana benthamiana; sgRNA; Begomovirus; vector; viruses

Published: September 30, 2020  Show citation

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Khan S, Mahmood MS, Rahman SU, Rizvi F, Ahmad A. Evaluation of the CRISPR/Cas9 system for the development of resistance against Cotton leaf curl virus in model plants. Plant Protect. Sci.. 2020;56(3):154-162. doi: 10.17221/105/2019-PPS.
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    References

    1. Adli M. (2018): The CRISPR tool kit for genome editing and beyond. Nature Communications, 9: 1911-1933. Go to original source... Go to PubMed...
    2. Ahmad A., Rahman Z., Hameed U., Rao A.Q., Ahad A., Yasmeen A., Akram F., Bajwa K.S., Sheffler J., Nasir I.A., Shahid A.A., Iqbal M.J., Husnain T., Haider M.S., Brown J.K. (2017): Engineered disease resistance in cotton using rna interference to knock down Cotton leaf curl Kokran virus-Burewala and Cotton leaf curl Multan betasatellite Expression. Viruses, 9: 257. doi:10.3390/v9090257 Go to original source... Go to PubMed...
    3. Ali Z., Ali S., Tashkandi M., Zaidi S.S., Mahfouz M.M. (2016): CRISPR/Cas9-Mediated immunity to geminiviruses: differential interference and evasion. Scientific Reports, 26: 6. doi: 10.1038/srep30223 Go to original source... Go to PubMed...
    4. Bortesi L., Fischer R. (2015): The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 33: 41-52. Go to original source... Go to PubMed...
    5. Briddon R.W., Markham P.G. (2000): Cotton leaf curl virus disease. Virology Research, 71: 151-159. Go to original source... Go to PubMed...
    6. Carthew R.W., Sontheimer E.J. (2009): Origins and mechanisms of miRNAs and siRNAs. Cell, 136: 642-655. Go to original source... Go to PubMed...
    7. Chatterji A., Chatterji U., Beachy R.N., Fauquet C.M. (2000): Sequence parameters that determine specificity of binding of the replication-associated protein to its cognate site in two strains of Tomato leaf curl Virus-New Delhi. Virology, 273: 341-350. Go to original source... Go to PubMed...
    8. Chen J., Zhang W., Lin J., Wang F., Wu M., Chen C., Zheng Y., Peng X., Li J., Yuan Z. (2014): An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases. Molecular Therapy, 22: 303-311. Go to original source... Go to PubMed...
    9. Czosnek H., Hariton-Shalev A., Sobol I., Gorovits R., Ghanim M. (2017): The incredible journey of begomoviruses in their whitefly vector. Viruses, 9: 273. doi: 10.3390/v910027 Go to original source... Go to PubMed...
    10. Fu Y., Foden J.A, Khayter C., Maeder M.L., Reyon D., Joung J.K., Sander J.D. (2013): High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 31: 822-826. Go to original source... Go to PubMed...
    11. Ha C., Coombs S., Revill P., Harding R., Vu M., Dale J. (2008). Molecular characterization of begomoviruses and DNA satellites from Vietnam: additional evidence that the New World geminiviruses were present in the Old World prior to continental separation. Journal of General Virology, 8: 312-326. Go to original source... Go to PubMed...
    12. Ishino Y., Krupovic M., Forterre P. (2018): History of CRISPRCas from encounter with a mysterious repeated sequence to genome editing technology. Journal of Bacteriology, 200: e00580-17. doi:10.1128/JB.00580-17 Go to original source... Go to PubMed...
    13. Ji X., Zhang H., Zhang Y., Wang Y., Gao C. (2015): Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants. Nature Plants, 28: 15144. doi: 10.1038/nplants.2015.144 Go to original source... Go to PubMed...
    14. Jiang W., Brueggeman A.J., Horken K.M., Plucinak T.M., Weeks D.P. (2014): Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryotic Cell, 13: 1465-1469. Go to original source... Go to PubMed...
    15. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. (2012): A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337: 816-821. Go to original source... Go to PubMed...
    16. Khan Z., Khan S.H., Ahmad A., Aslam S., Mubarik M.S., Khan S. (2018): CRISPR/dCas9-mediated inhibition of replication of Begomoviruses. International Journal of Agriculture and Biology, 21: 711-718.
    17. Kirthi N., Priyadarshini C.G., Sharma P., Maiya S.P., Hemalatha V., Sivaraman P., Dhawan P., Rishi N., Savithri H.S. (2004): Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India. Archives of Virology, 149: 2047-2057. Go to PubMed...
    18. Leuzinger K., Dent M., Hurtado J., Stahnke J., Lai H., Zhou X., Chen Q. (2013): Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. Journal of Visual Experiments, 23: 77. doi: 10.3791/50521 Go to original source... Go to PubMed...
    19. Long L., Guo D.D., Gao W., Yang W.W., Hou L.P., Ma X.N., Miao Y.C., Botella J.R., Song C.P. (2018): Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression. Plant Methods, 14: 85-93. Go to original source... Go to PubMed...
    20. Mali P., Yang L., Esvelt K.M, Aach J., Guell M., DiCarlo J.E., Church G.M. (2013): RNA-guided human genome engineering via Cas9. Science, 339: 823-826. Go to original source... Go to PubMed...
    21. Mansoor S., Khan S.H., Bashir A., Saeed M., Zafar Y., Malik K.A., Briddon R., Stanley J., Markham P.G. (1999): Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan. Virology, 259: 190-199. Go to original source... Go to PubMed...
    22. Masood M., Briddon R.W. (2018): Transmission of cotton leaf curl disease: answer to a long-standing question. Virology General, 54: 743-745. Go to original source... Go to PubMed...
    23. Mohanta T., Bashir T., Hashem A., AbdAllah E., Bae H. (2017): Genome editing tools in plants. Genes (Basel), 8: 399-424. Go to original source... Go to PubMed...
    24. Mubarik S.M., Khan S.H., Sadia B., Ahmad A. (2019): CRISPR-Cas9 based suppression of Cotton leaf curl Virus in Nicotiana benthamiana. International Journal of Agriculture and Biology, 3: 517-522.
    25. 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: 1120-1123. Go to original source... Go to PubMed...
    26. Qadir R., Khan Z.A., Monga D., Khan J.A. (2019): Diversity and Recombination analysis of Cotton leaf curl Multan virus: a highly emerging begomovirus in northern India. BMC Genomics, 20: 274. doi: 10.1186/s12864-019-5640-2 Go to original source... Go to PubMed...
    27. Rahman M.U., Khan A.Q., Rahmat Z., Iqbal M.A., Zafar Y. (2017): Genetics and genomics of cotton leaf curl disease, its viral causal agents and whitefly vector: a way forward to sustain cotton fiber security. Frontiers in Plant Science, 8: 1157-1168. Go to original source... Go to PubMed...
    28. Rojas M.R., Hagen C., Lucas W.J., Gilbertson R.L. (2005): Exploiting chinks in the plant's armor: evolution and emergence of geminiviruses. Annual Review of Phytopathology, 43: 361-394. Go to original source... Go to PubMed...
    29. Saeed M., Briddon R.W., Dalakourous A., Krczal G., Wasseneger M. (2015): Functional analysis of Cotton leaf curl kokran virus/cotton leaf curl Multan betasatellite RNA silencing suppressors. Biology, 4: 697-714. Go to original source... Go to PubMed...
    30. Sattar M.N., Kvarnheden A., Saeed M., Briddon R.W. (2013): Cotton leaf curl disease - an emerging threat to cotton production worldwide. Journal of General Virology, 94: 695-710. Go to original source... Go to PubMed...
    31. Sovová T., Kerins G., Demnerová K., Ovesná J. (2017): Genome editing with engineered nucleases in economically important animals and plants: state of the art in the research pipeline. Current Issues in Molecular Biology, 21: 41-62. Go to PubMed...
    32. Tahir M.N., Amin I., Briddon R.W., Mansoor S. (2011): The merging of two dynasties-identification of an African cotton leaf curl disease-associated begomovirus with cotton in Pakistan. PLoS ONE, 6:(5)e20366. doi: 10.1371/journal.pone.0020366 Go to original source... Go to PubMed...
    33. Wei J., He Y.Z., Guo Q., Guo T., Liu Y.Q., Zhou X.P., Liu S.S. Wang W.X. (2017): Vector development and vitellogenin determine the transovarial transmission of begomoviruses, Protocols of National Academy of Sciences USA, 114: 6746-6751. Go to original source... Go to PubMed...
    34. Xing H.L., Dong L., Wang Z.P., Zhang H.Y., Han C.Y., Liu B., Wang X.C., Chen Q.J. (2014): A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology, 14: 327. doi:10.1186/s12870-014-0327-y Go to original source... Go to PubMed...
    35. Yin K., Han T., Xie K., Zhao J., Song J., Liu Y. (2019): Engineer complete resistance to Cotton Leaf Curl Multan virus by the CRISPR/Cas9 system in Nicotiana Benthamiana. Phytopathology Research, 1:9. doi: 10.1186/s42483-019-0017-7 Go to original source...
    36. Zaidi S.S., Tashkandi M., Mansoor S., Mahfouz M.M. (2016): Engineering plant immunity using crispr/cas9 to generate virus resistance. Frontiers in Plant Science, 7: 1673. doi: 10.3389/fpls.2016.01673. Go to original source... Go to PubMed...
    37. Zhang Z., Mao Y., Ha S., Liu W., Botella J,R., Zhu J.K. (2016): A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Reports, 35: 1519-1533. Go to original source... Go to PubMed...

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