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Efficient CRISPR/Cas9-mediated genome editing in Rehmannia glutinosa

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Here, we cloned a phytoene desaturase (PDS) gene from Rehmannia glutinosa, and realized RgPDS1 knock out in R. glutinosa resulted in the generation of albino plants.

Abstract

Rehmannia glutinosa is a highly important traditional Chinese medicine (TCM) with specific pharmacology and economic value. R. glutinosa is a tetraploid plant, to date, no report has been published on gene editing of R. glutinosa. In this study, we combined the transcriptome database of R. glutinosa and the reported phytoene desaturase (PDS) gene sequences to obtain the PDS gene of R. glutinosa. Then, the PDS gene was used as a marker gene to verify the applicability and gene editing efficiency of the CRISPR/Cas9 system in R. glutinosa. The constructed CRISPR/Cas9 system was mediated by Agrobacterium to genetically transform into R. glutinosa, and successfully regenerated fully albino and chimeric albino plants. The next-generation sequencing (NGS) confirmed that the albino phenotype was indeed caused by RgPDS gene target site editing, and it was found that base deletion was more common than insertion or replacement. Our results revealed that zCas9 has a high editing efficiency on the R. glutinosa genome. This research lays a foundation for further use of gene editing technology to study the molecular functions of genes, create excellent germplasm, accelerate domestication, and improve the yield and quality of R. glutinosa.

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References

  • Alagoz Y, Gurkok T, Zhang BH, Unver T (2016) Manipulating the biosynthesis of bioactive compound alkaloids for next-generation metabolic engineering in Opium poppy using CRISPR-Cas 9 genome editing technology. Sci Rep 6:30910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Banakar R, Schubert M, Collingwood M, Vakulskas C, Eggenberger AL, Wang K (2020) Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS) Gene. Rice 13:4

    Article  PubMed  PubMed Central  Google Scholar 

  • Brausemann A, Gemmecker S, Koschmieder J, Ghisla S, Beyer P, Einsle O (2017) Structure of phytoene desaturase provides insights into herbicide binding and reaction mechanisms involved in carotene desaturation. Structure 25:1222–1232

    Article  CAS  PubMed  Google Scholar 

  • Breitler JC, Dechamp E, Campa C, Rodrigues LAZ, Guyot R, Marraccini P, Etienne H (2018) CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of Coffea canephora. Plant Cell Tiss Org 134:383–394

    Article  CAS  Google Scholar 

  • Charrier A, Vergne E, Dousset N, Richer A, Petiteau A, Chevreau E (2019) Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPR-Cas9 system. Front Plant Sci 10:40

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen K, Wang Y, Zhang R, Zhang H, Gao C (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol 70:667–697

    Article  CAS  PubMed  Google Scholar 

  • Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, Cole MA, Liu DR, Joung JK, Bauer DE, Pinello L (2019) CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37(3):224–226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Do PT, Nguyen CX, Bui HT, Tran LTN, Stacey G, Gillman JD, Zhang ZJ, Stacey MG (2019) Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and alpha-linolenic acid phenotype in soybean. BMC Plant Biol 19:311

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Fan D, Liu TT, Li CF, Jiao B, Li S, Hou YS, Luo KM (2015) Efficient CRISPR/Cas9-mediated targeted mutagenesis in populus in the first generation. Sci Rep 5:12217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gaj T, Gersbach CA, Barbas CF (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gudu S, Procunier JD, Ziauddin A, Kasha KJ (1993) Anther culture derived homozygous lines in Hordeum bulbosum. Plant Breed 110:109–115

    Article  Google Scholar 

  • Gupta A, Hall VL, Kok FO, Shin M, McNulty JC, Lawson ND, Wolfe SA (2013) Targeted chromosomal deletions and inversions in zebrafish. Genome Res 23:1008–1017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Haeussler M, Schonig K, Eckert H, Eschstruth A, Mianne J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17(1):148

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Huang TK, Puchta H (2019) CRISPR/Cas-mediated gene targeting in plants: finally a turn for the better for homologous recombination. Plant Cell Rep 38:443–453

    Article  CAS  PubMed  Google Scholar 

  • Kaur N, Alok A, Shivani KN, Pandey P, Awasthi P, Tiwari S (2018) CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali Genome. Funct Integr Genomics 18:89–99

    Article  CAS  PubMed  Google Scholar 

  • Kelliher T, Starr D, Richbourg L, Chintamanani S, Delzer B, Nuccio ML, Green J, Chen Z, McCuiston J, Wang W, Liebler T, Bullock P, Martin B (2017) MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature 542:105–109

    Article  CAS  PubMed  Google Scholar 

  • Klimek-Chodacka M, Oleszkiewicz T, Lowder LG, Qi Y, Baranski R (2018) Efficient CRISPR/Cas9-based genome editing in carrot cells. Plant Cell Rep 37:575–586

    Article  CAS  PubMed  PubMed Central  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  PubMed  PubMed Central  Google Scholar 

  • Li MJ, Yang YH, Li XY, Gu L, Wang FJ, Feng FJ, Tian YH, Wang FQ, Wang XR, Lin WX, Chen XJ, Zhang ZY (2015) Analysis of integrated multiple “omics” datasets reveals the mechanisms of initiation and determination in the formation of tuberous roots in Rehmannia glutinosa. J Exp Bot 66:5837–5851

    Article  CAS  PubMed  Google Scholar 

  • Li T, Yang X, Yu Y, Si X, Zhai X, Zhang H, Dong W, Gao C, Xu C (2018) Domestication of wild tomato is accelerated by genome editing. Nat Biotechnol 36:1211–1216

    Article  CAS  Google Scholar 

  • Li ZH, Matthews PD, Burr B, Wurtzel ET (1996) Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway. Plant Mol Biol 30:269–279

    Article  CAS  PubMed  Google Scholar 

  • Liu C, Li X, Meng D, Zhong Y, Chen C, Dong X, Xu X, Chen B, Li W, Li L, Tian X, Zhao H, Song W, Luo H, Zhang Q, Lai J, Jin W, Yan J, Chen S (2017) A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates haploid induction in Maize. Mol Plant 10:520–522

    Article  CAS  PubMed  Google Scholar 

  • Liu TT, Fan D, Ran LY, Jiang YZ, Liu R, Luo JM (2015) Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus. Hereditas 37:1044–1052

    CAS  PubMed  Google Scholar 

  • Lv J, Yu K, Wei J, Gui H, Liu C, Liang D, Wang Y, Zhou H, Carlin R, Rich R, Lu T, Que Q, Wang WC, Zhang X, Kelliher T (2020) Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3. Nat Biotechnol 38:1397–1401

    Article  CAS  PubMed  Google Scholar 

  • Ma CF, Liu MC, Li QF, Si J, Ren XS, Song HY (2019) Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Hortic Res 5:164–169

    Google Scholar 

  • Ma X, Zhu Q, Chen Y, Liu YG (2016) CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9:961–974

    Article  CAS  PubMed  Google Scholar 

  • Mercx S, Tollet J, Magy B, Navarre C, Boutry M (2016) Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells. Front Plant Sci 7:40

    Article  PubMed  PubMed Central  Google Scholar 

  • Nishitani C, Hirai N, Komori S, Wada M, Okada K, Osakabe K, Yamamoto T, Osakabe Y (2016) Efficient genome editing in apple using a CRISPR/Cas9 system. Sci Rep 6:31481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Odipio J, Alicai T, Ingelbrecht I, Nusinow DA, Bart R (2017) Taylor NJ (2017) Efficient CRISPR/Cas9 genome editing of Phytoene desaturase in cassava. Front Plant Sci 8:1780

    Article  PubMed  PubMed Central  Google Scholar 

  • Park J, Lim K, Kim JS, Bae S (2017) Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33(2):286–288

    Article  CAS  PubMed  Google Scholar 

  • Qi J, Li X, Song J, Eneji AE, Ma X (2008) Genetic relationships among Rehmannia glutinosa cultivars and varieties. Planta Med 74(15):1846–1852

    Article  CAS  PubMed  Google Scholar 

  • Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17(5):471–482

    Article  CAS  PubMed  Google Scholar 

  • Ren FR, Ren C, Zhang Z, Duan W, Lecourieux D, Li SH, Liang ZC (2019) Efficiency optimization of CRISPR/Cas9-mediated targeted mutagenesis in grape. Front Plant Sci 10:612

    Article  PubMed  PubMed Central  Google Scholar 

  • Shen RX, Wang L, Liu XP, Wu J, Jin WW, Zhao XC, Xie XR, Zhu QL, Tang HW, Li Q, Chen LT, Liu YG (2017) Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice. Nat Commun 8:1310

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Shi M, Gong H, Cui LJ, Wang Q, Wang C, Wang Y, Kai GY (2020) Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots. Ind Crop Prod 148:112277

    Article  CAS  Google Scholar 

  • Tian SW, Jiang LJ, Gao Q, Zhang J, Zong M, Zhang HY, Ren Y, Guo SG, Gong GY, Liu F, Xu Y (2017a) Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Rep 36:399–406

    Article  CAS  PubMed  Google Scholar 

  • Tian YH, Feng FJ, Zhang B, Li MJ, Wang FQ, Gu L, Chen AG, Li ZJ, Shan WB, Wang XR, Chen XJ, Zhang ZY (2017b) Transcriptome analysis reveals metabolic alteration due to consecutive monoculture and abiotic stress stimuli in Rehamannia glutinosa Libosch. Plant Cell Rep 36:859–875

    Article  CAS  PubMed  Google Scholar 

  • Wang FQ, Zhi JY, Zhang ZY, Wang LN, Suo YF, Xie CX, Li MJ, Zhang B, Du JF, Gu L, Sun HZ (2017) Transcriptome analysis of salicylic acid treatment in Rehmannia glutinosa hairy roots using RNA-seq technique for identification of genes involved in acteoside biosynthesis. Front Plant Sci 8:787

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang Y (2003) S-RNase-mediated self-incompatibility. J Exp Bot 54:115–122

    Article  CAS  PubMed  Google Scholar 

  • Wilson FM, Harrison K, Armitage AD, Simkin AJ, Harrison RJ (2019) CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Plant Methods 15:45

    Article  PubMed  PubMed Central  Google Scholar 

  • Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xu ZS, Feng K, Xiong AS (2019) CRISPR/Cas9-mediated multiply targeted mutagenesis in orange and purple carrot plants. Mol Biotechnol 61:191–199

    Article  CAS  PubMed  Google Scholar 

  • Yao L, Zhang Y, Liu C, Liu Y, Wang Y, Liang D, Liu J, Sahoo G, Kelliher T (2018) OsMATL mutation induces haploid seed formation in indica rice. Nat Plants 4:530–533

    Article  CAS  PubMed  Google Scholar 

  • Ye MW, Peng Z, Tang D, Yang ZM, Li DW, Xu YM, Zhang CZ, Huang SW (2018) Generation of self-compatible diploid potato by knockout of S-RNase. Nat Plants 4:651–654

    Article  CAS  PubMed  Google Scholar 

  • Zhang RX, Li MX, Jia ZP (2008) Rehmannia glutinosa: review of botany, chemistry and pharmacology. J Ethnopharmacol 117:199–214

    Article  CAS  PubMed  Google Scholar 

  • Zhang X, Xu G, Cheng C, Lei L, Sun J, Xu Y, Deng C, Dai Z, Yang Z, Chen X, Liu C, Tang Q, Su J (2021) Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Hemp (Cannabis sativa L.). Plant Biotechnol J. https://doi.org/10.1111/pbi.13611

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D (2017) Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. Plant J 91:714–724

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • Zhi JY, Li YJ, Zhang ZY, Yang CF, Geng XT, Zhang M, Li XR, Zuo X, Li MJ, Huang Y, Wang FQ, Xie CX (2018) Molecular regulation of catalpol and acteoside accumulation in radial striation and non-radial striation of Rehmannia glutinosa tuberous root. Int J Mol Sci 19:3751

    Article  PubMed Central  CAS  Google Scholar 

  • Zhong Y, Chen BJ, Li MR, Wang D, Jiao YY, Qi XL, Wang M, Liu ZK, Chen C, Wang YW, Chen M, Li JL, Xiao ZJ, Cheng DH, Liu WX, Boutilier K, Liu CX, Chen SJ (2020) A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis. Nat Plants 6(5):466–472

    Article  CAS  PubMed  Google Scholar 

  • Zhou Z, Tan H, Li Q, Chen J, Gao S, Wang Y, Chen W, Zhang L (2018) CRISPR/Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza. Phytochemistry 148:63–70

    Article  PubMed  CAS  Google Scholar 

  • Zhu HC, Li C, Gao CX (2020) Applications of CRISPR-Cas in agriculture and plant biotechnology. Nat Rev Mol Cell Bio 21:661–677

    Article  CAS  Google Scholar 

  • Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C (2017) Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35:438–440

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr. Qijun Chen for providing the pKSE401 vector. This project was supported by the National Science Foundation of China (NSFC Grant Nos. 81872950 & 82073952)

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Author notes

  1. Xinrong Li, Xin Zuo and Mingming Li have contributed equally to this research work.

Authors and Affiliations

  1. College of Agronomy, Henan Agricultural University, Zhengzhou, 450046, China

    Xinrong Li, Xin Zuo, Mingming Li, Xu Yang, Jingyu Zhi, Hongzheng Sun & Fengqing Wang

  2. School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, 450046, China

    Caixia Xie

  3. College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Zhongyi Zhang

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  1. Xinrong Li
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  2. Xin Zuo
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Contributions

FW conceived the project, designed the experiments. XL, XZ, ML, XY and JZ performed experiments. FW, XL, XZ and ML prepared the manuscript together. HS, CX and ZZ participated in data analysis.

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Correspondence to Fengqing Wang.

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Communicated by Yiping Qi.

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Li, X., Zuo, X., Li, M. et al. Efficient CRISPR/Cas9-mediated genome editing in Rehmannia glutinosa. Plant Cell Rep 40, 1695–1707 (2021). https://doi.org/10.1007/s00299-021-02723-3

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