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Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations

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Abstract

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We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy.

Abstract

CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.

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References

  • Bove JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 88:7–37

    Google Scholar 

  • Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytasa DF (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217

    Article  CAS  Google Scholar 

  • Chen KL, Wang YP, Zhang R, Zhang HW, Gao CX (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. In: Merchant SS (ed) Annual review of plant biology, vol 70, pp. 667–697. Annual Reviews, Palo Alto

  • Dang Y, Jia GX, Choi J, Ma HM, Anaya E, Ye CT, Shankar P, Wu HQ (2015) Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol 16:1–10

    Article  CAS  Google Scholar 

  • Graf R, Li X, Chu VT, Rajewsky K (2019) sgRNA sequence motifs blocking efficient CRISPR/Cas9-mediated gene editing. Cell Rep 26:1098

    Article  CAS  Google Scholar 

  • He XB, Wang YF, Yang FY, Wang B, Xie HH, Gu LK, Zhao TY, Liu XX, Zhang DB, Ren QW, Liu XY, Liu Y, Gao CX, Gu F (2019) Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA(Gln)-processing system in human cells. J Biol Chem 294:9308–9315

    Article  Google Scholar 

  • Hu Y, Zhang JL, Jia HG, Sosso D, Li T, Frommer WB, Yang B, White FF, Wang NA, Jones JB (2014) Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proc Natl Acad Sci USA 111:E521–E529

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Jia HG, Zhang YZ, Orbovic V, Xu J, White FF, Jones JB, Wang N (2017) Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol J 15:817–823

    Article  CAS  Google Scholar 

  • Jia H, Wang N (2020) Generation of homozygous canker‐resistant citrus in the T0 generation using CRISPR‐SpCas9p, Plant Biotechnol J

  • Jia H, Zou X, Orbovic V, Wang N (2019) Genome editing in Citrus Tree with CRISPR/Cas9, Plant genome editing with CRISPR systems, pp 235–241

  • 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  Google Scholar 

  • Li JF, Norville JE, Aach J, McCormack M, Zhang DD, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691

    Article  CAS  Google Scholar 

  • Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261

    Article  CAS  Google Scholar 

  • Lin CS, Hsu CT, Yang LH, Lee LY, Fu JY, Cheng QW, Wu FH, Hsiao HCW, Zhang YS, Zhang R, Chang WJ, Yu CT, Wang W, Liao LJ, Gelvin SB, Shih MC (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16:1295–1310

    Article  CAS  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 

  • Nekrasov V, Staskawicz B, Weigel D, Jones JDG, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693

    Article  CAS  Google Scholar 

  • Omar AA, Dutt M, Gmitter FG, Grosser JW (2016) Somatic embryogenesis: still a relevant technique in citrus improvement. Methods Mol Biol 1359:289–327

    Article  CAS  Google Scholar 

  • Omura M, Shimada T (2016) Citrus breeding, genetics and genomics in Japan. Breed Sci 66:3–17

    Article  CAS  Google Scholar 

  • Peng AH, Chen SC, Lei TG, Xu LZ, He YR, Wu L, Yao LX, Zou XP (2017) Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnol J 15:1509–1519

    Article  CAS  Google Scholar 

  • Port F, Bullock SL (2016) Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods 13:852

    Article  CAS  Google Scholar 

  • Puchta H (2017) Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol 36:1–8

    Article  CAS  Google Scholar 

  • Qi YP, Li XH, Zhang Y, Starker CG, Baltes NJ, Zhang F, Sander JD, Reyon D, Joung JK, Voytas DF (2013) Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases. G3-Genes Genomes Ge 3:1707–1715

    Google Scholar 

  • Qi WW, Zhu T, Tian ZR, Li CB, Zhang W, Song RT (2016) High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol 16:8

    Article  Google Scholar 

  • Schindele P, Wolter F, Puchta H (2018) Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13. FEBS Lett 592:1954–1967

    Article  CAS  Google Scholar 

  • Shan QW, Wang YP, Li J, Zhang Y, Chen KL, Liang Z, Zhang K, Liu JX, Xi JJ, Qiu JL, Gao CX (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688

    Article  CAS  Google Scholar 

  • Shiraki T, Kawakami K (2018) A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish. Sci Rep 8:1–14

    Article  Google Scholar 

  • Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM (2016) Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun 7:1–7

    Article  Google Scholar 

  • Tang X, Ren QR, Yang LJ, Bao Y, Zhong ZH, He Y, Liu SS, Qi CY, Liu BL, Wang Y, Sretenovic S, Zhang YX, Zheng XL, Zhang T, Qi YP, Zhang Y (2019) Single transcript unit CRISPR 20 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnol J 17:1431–1445

    Article  CAS  Google Scholar 

  • Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided Fokl nucleases for highly specific genome editing. Nat Biotechnol 32:569

    Article  CAS  Google Scholar 

  • Waibel F, Filipowicz W (1990) U6 SNRNA genes of arabidopsis are transcribed by RNA polymerase-III but contain the same 2 upstream promoter elements as RNA polymerase-II-transcribed U-SNRNA genes. Nucleic Acids Res 18:3451–3458

    Article  CAS  Google Scholar 

  • Wang N (2019) The citrus Huanglongbing crisis and potential solutions. Mol Plant 12:607–609

    Article  CAS  Google Scholar 

  • Wang N, Pierson EA, Setubal JC, Xu J, Levy JG, Zhang YZ, Li JY, Rangel LT, Martins J (2017) The Candidatus liberibacter-host interface: insights into pathogenesis mechanisms and disease control. Annu Rev Phytopathol 55(55):451–482

    Article  CAS  Google Scholar 

  • Wang ZP, Wang SB, Li DW, Zhang Q, Li L, Zhong CH, Liu YF, Huang HW (2018) Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol J 16:1424–1433

    Article  CAS  Google Scholar 

  • Weeks DP (2017) Gene editing in polyploid crops: wheat, camelina, canola, potato, cotton, peanut, sugar cane, and citrus. Prog Mol Biol Transl Sci 149:65–80

    Article  CAS  Google Scholar 

  • Woo JW, Kim J, Il Kwon S, Corvalan C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–U156

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Xu L, Zhao LX, Gao YD, Xu J, Han RZ (2017) Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA. Nucleic Acids Res 45:e28

    PubMed  Google Scholar 

  • Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Article  CAS  Google Scholar 

  • Zhang Y, Liang Z, Zong Y, Wang YP, Liu JX, Chen KL, Qiu JL, Gao CX (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:1–8

    Google Scholar 

  • Zhang F, LeBlanc C, Irish VF, Jacob Y (2017) Rapid and efficient CRISPR/Cas9 gene editing in Citrus using the YAO promoter. Plant Cell Rep

  • Zhang YP, Wang J, Wang ZB, Zhang YM, Shi SB, Nielsen J, Liu ZH (2019a) A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat Commun 10:1–10

    Article  Google Scholar 

  • Zhang YX, Malzahn AA, Sretenovic S, Qi YP (2019b) The emerging and uncultivated potential of CRISPR technology in plant science. Nat Plants 5:778–794

    Article  Google Scholar 

Download references

Acknowledgements

The research has been supported by USDA National Institute of Food and Agriculture grant # 2018-70016-27412, #2016-70016-24833, and #2019-70016-29796, USDA-NIFA Plant Biotic Interactions Program 2017-67013-26527, Florida Citrus Initiative, and Florida Citrus Research and Development Foundation.

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Authors and Affiliations

  1. Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA

    Xiaoen Huang, Yuanchun Wang, Jin Xu & Nian Wang

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  1. Xiaoen Huang
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  2. Yuanchun Wang
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  3. Jin Xu
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  4. Nian Wang
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Contributions

XH and NW designed the study, analyzed the data and wrote the manuscript. YW conducted plant transformation. JX was involved in citrus genome sequence analysis.

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Correspondence to Nian Wang.

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Huang, X., Wang, Y., Xu, J. et al. Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations. Plant Mol Biol 104, 297–307 (2020). https://doi.org/10.1007/s11103-020-01043-6

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