Skip to main content
SpringerLink
Log in

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene

  • Brief Report
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both ‘Carrizo’ citrange and ‘Duncan’ grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

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

Similar content being viewed by others

References

  • Ballester A, Cervera M, Peña L (2008) Evaluation of selection strategies alternative to nptII in genetic transformation of citrus. Plant Cell Rep 27:1005–1015

    Article  CAS  PubMed  Google Scholar 

  • Blomme J, Develtere W, Köse A, Arraiza Ribera J, Brugmans C, Jaraba-Wallace J, Decaestecker W, Rombaut D, Baekelandt A, Daniel Fernández Fernández Á (2022) The heat is on: a simple method to increase genome editing efficiency in plants. BMC Plant Biol 22:142

    Article  PubMed  PubMed Central  Google Scholar 

  • Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 1:7–37

    Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi M, Yun J-Y, Kim J-H, Kim J-S, Kim S-T (2021) The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana. Sci Rep 11:8087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Classic Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  Google Scholar 

  • Conti G, Xoconostle-Cázares B, Marcelino-Pérez G, Hopp HE, Reyes CA (2021) Citrus genetic transformation: an overview of the current strategies and insights on the new emerging technologies. Front Plant Sci 12:2519

    Article  Google Scholar 

  • Dominguez MM, Padilla CS, Mandadi KK (2022) A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars. Front Plant Sci. https://doi.org/10.3389/fpls.2022.878335

    Article  PubMed  PubMed Central  Google Scholar 

  • Doyle JJ, Doyle JL, Doyle JA, Doyle FJ (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Dutt M, Mou Z, Zhang X, Tanwir SE, Grosser JW (2020) Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures. BMC Biotechnol 20:1–7

    Article  Google Scholar 

  • Ferrarezi RS, Rodriguez K, Sharp D (2020) How historical trends in Florida all-citrus production correlate with devastating hurricane and freeze events. Weather 75:77–83

    Article  Google Scholar 

  • Gabriel D, Gottwald TR, Lopes SA, Wulff NA (2020) Bacterial pathogens of citrus: Citrus canker, citrus variegated chlorosis and Huanglongbing. The genus citrus. Elsevier, pp 371–389. https://doi.org/10.1016/B978-0-12-812163-4.00018-8

    Chapter  Google Scholar 

  • Gottwald TR, Graham JH (2014) Citrus diseases with global ramifications including citrus canker and huanglongbing. CABI Rev 2014:1–11

    Article  Google Scholar 

  • Grützner R, Martin P, Horn C, Mortensen S, Cram EJ, Lee-Parsons CWT, Stuttmann J, Marillonnet S (2021) High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns. Plant Commun. 2:100135

    Article  PubMed  Google Scholar 

  • Haque S, Hammami A, Xia T, Guan Z (2023) Market power in the florida orange juice processing industry

  • Harper SJ, Cowell SJ (2016) The past and present status of Citrus tristeza virus in Florida. J Citrus Pathol. https://doi.org/10.5070/C431032387

    Article  Google Scholar 

  • Huang K-M, Guan Z, Hammami A (2022a) The US fresh fruit and vegetable industry: An overview of production and trade. Agriculture 12:1719

    Article  Google Scholar 

  • Huang X, Wang Y, Wang N (2022b) Highly efficient generation of canker-resistant sweet orange enabled by an improved CRISPR/Cas9 system. Front Plant Sci 12:769907

    Article  PubMed  PubMed Central  Google Scholar 

  • Jia H, Wang N (2014) Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS ONE 9:e93806

    Article  PubMed  PubMed Central  Google Scholar 

  • Jia H, Orbović V, Wang N (2019) CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnol J 17:1928–1937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang C, Geng L, Wang J, Liang Y, Guo X, Liu C, Zhao Y, Jin J, Liu Z, Mu Y (2023) Multiplexed gene engineering based on dCas9 and gRNA-tRNA array encoded on single transcript. Int J Mol Sci 24:8535

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • LeBlanc C, Zhang F, Mendez J, Lozano Y, Chatpar K, Irish VF, Jacob Y (2018) Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J 93:377–386

    Article  CAS  PubMed  Google Scholar 

  • Li R, Liu C, Zhao R, Wang L, Chen L, Yu W, Zhang S, Sheng J, Shen L (2019) CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance. BMC Plant Biol 19:1–13

    Google Scholar 

  • Lu Y, Tian Y, Shen R, Yao Q, Zhong D, Zhang X, Zhu J (2021) Precise genome modification in tomato using an improved prime editing system. Plant Biotechnol J 19:415

    Article  PubMed  Google Scholar 

  • Milner MJ, Craze M, Hope MS, Wallington EJ (2020) Turning up the temperature on CRISPR: increased temperature can improve the editing efficiency of wheat using CRISPR/Cas9. Front Plant Sci 11:1780

    Article  Google Scholar 

  • Minkenberg B, Wheatley M, Yang YN (2017) CRISPR/Cas9-enabled multiplex genome editing and its application. Gene Editing Plants 149:111–132

    Article  CAS  Google Scholar 

  • Omar AA, Murata MM, El-Shamy HA, Graham JH, Grosser JW (2018) Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice. Trans Res 27:179–191

    Article  CAS  Google Scholar 

  • Orbović V, Grosser JW (2006) Citrus. Agrobacterium Protocols-Methods Molecul Biol 2:245–257

    Google Scholar 

  • Orbović V, Grosser JW (2015) Citrus transformation using juvenile tissue explants. In: Wang K (ed) Agrobacterium Protocols. Springer, New York, New York, pp 245–257

    Chapter  Google Scholar 

  • Orbović V, Ravanfar SA, Acanda Y, Merritt BA, Levy A, Narvaez J, Lovatt C (2021) Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp. Trans Res 30:687–699

    Article  Google Scholar 

  • Peng A, Zhang J, Zou X, He Y, Xu L, Lei T, Yao L, Li Q, Chen S (2021) Pyramiding the antimicrobial PR1aCB and AATCB genes in “Tarocco” blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance. Trans Res 30:635–647

    Article  CAS  Google Scholar 

  • Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu L-J (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 

  • Rai MK, Shekhawat NS (2014) Recent advances in genetic engineering for improvement of fruit crops. Plant Cell, Tissue, Organ Cult 116:1–15

    Article  CAS  Google Scholar 

  • Schindele A, Gehrke F, Schmidt C, Röhrig S, Dorn A, Puchta H (2022) Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats. Nat Commun 13:1502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Singerman A, Rogers ME (2020) The economic challenges of dealing with citrus greening: the case of Florida. J Integr Pest Manag 11:3

    Article  Google Scholar 

  • Stuttmann J, Barthel K, Martin P, Ordon J, Erickson JL, Herr R, Ferik F, Kretschmer C, Berner T, Keilwagen J (2021) Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants. Plant J 106:8–22

    Article  CAS  PubMed  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 112:3570–3575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang F, LeBlanc C, Irish VF et al (2017) Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter. Plant Cell Rep 36:1883–1887. https://doi.org/10.1007/s00299-017-2202-4

  • Zhang Y, Wang J, Wang Z, Zhang Y, Shi S, Nielsen J, Liu Z (2019) A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10:1053

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by the USDA/NIFA Emergency Citrus Disease Research and Extension Program.

Author information

Authors and Affiliations

  1. Citrus Research and Education Center, University of Florida, Lake Alfred, FL, 33850, USA

    Poulami Sarkar, Jorge Santiago Vazquez, Amit Levy & Vladimir Orbović

  2. Department of Cell Sciences and Microbiology, University of Florida, Gainesville, FL, 32611, USA

    Amit Levy

  3. Department of Plant Pathology, University of Florida, Gainesville, FL, 32602, USA

    Mingxi Zhou & Zhonglin Mou

  4. Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32611, USA

    Mingxi Zhou

Authors
  1. Poulami Sarkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jorge Santiago Vazquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mingxi Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amit Levy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhonglin Mou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vladimir Orbović
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

ZM, PS, and VO designed experiments. PS, JSV, and VO performed experiments. MZ and ZM constructed binary vector. PS and VO wrote the manuscript. PS, AL, and ZM edited the manuscript. All authors agreed with the content of the manuscript.

Corresponding author

Correspondence to Vladimir Orbović.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

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 15 KB)

Supplementary file2 (DOCX 19 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

Sarkar, P., Santiago Vazquez, J., Zhou, M. et al. Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene. Transgenic Res 33, 59–66 (2024). https://doi.org/10.1007/s11248-024-00380-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-024-00380-2

Keywords

Search

Navigation