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Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

Science China Life Sciences volume 60, pages 506–515 (2017)Cite this article

An Erratum to this article was published on 09 August 2019

This article has been updated

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.

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Change history

  • 09 August 2019

    The same figure was misused for the PCR/RE assay results of Gn1a and GW2 fragments in Figure 3, and the arrows in the graphicsal result of GW2 were not on the tape. The corrected Figure 3 is as follows.

  • 09 August 2019

    The same figure was misused for the PCR/RE assay results of Gn1a and GW2 fragments in Figure 3, and the arrows in the graphicsal result of GW2 were not on the tape. The corrected Figure 3 is as follows.

References

  • Ashikari, M., Sakakibara, H., Lin, S., Yamamoto, T., Takashi, T., Nishimura, A., Angeles, E.R., Qian, Q., Kitano, H., and Matsuoka, M. (2005). Cytokinin oxidase regulates rice grain production. Science 309, 741–745.

    CAS  Article  PubMed  Google Scholar 

  • Casini, A., Storch, M., Baldwin, G.S., and Ellis, T. (2015). Bricks and blueprints: methods and standards for DNA assembly. Nat Rev Mol Cell Biol 16, 568–576.

    CAS  Article  PubMed  Google Scholar 

  • Chen, S., Yang, Y., Shi, W., Ji, Q., He, F., Zhang, Z., Cheng, Z., Liu, X., and Xu, M. (2008). Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance. Plant Cell 20, 1850–1861.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Cho, Y.G., Eun, M.Y., McCouch, S.R., and Chae, Y.A. (1994). The semidwarf gene, sd-1, of rice (Oryza sativa L.). II. molecular mapping and marker-assisted selection. Theoret Appl Genet 89, 54.

    CAS  Google Scholar 

  • Doench, J.G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E.W., Donovan, K.F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H.W., Listgarten, J., and Root, D.E. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34, 184–191.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Fan, C., Xing, Y., Mao, H., Lu, T., Han, B., Xu, C., Li, X., and Zhang, Q. (2006). GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor Appl Genet 112, 1164–1171.

    CAS  Article  PubMed  Google Scholar 

  • Fukuma, M., Ganmyo, Y., Miura, O., Ohyama, T., and Shimizu, N. (2016). Cloning and characterization of a human genomic sequence that alleviates repeat-induced gene silencing. PLoS One 11, e0153338.

    Article  PubMed  PubMed Central  Google Scholar 

  • Generoso, W.C., Gottardi, M., Oreb, M., and Boles, E. (2016). Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. J Microbiol Methods 127, 203–205.

    CAS  Article  PubMed  Google Scholar 

  • Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6, 271–282.

    CAS  Article  PubMed  Google Scholar 

  • Hu, X., Wang, C., Fu, Y., Liu, Q., Jiao, X., and Wang, K. (2016). Expanding the range of CRISPR/Cas9 genome editing in rice. Mol Plant 9, 943–945.

    CAS  Article  PubMed  Google Scholar 

  • Huang, X., Qian, Q., Liu, Z., Sun, H., He, S., Luo, D., Xia, G., Chu, C., Li, J., and Fu, X. (2009). Natural variation at the DEP1 locus enhances grain yield in rice. Nat Genet 41, 494–497.

    CAS  Article  PubMed  Google Scholar 

  • Jiang, L., Liu, X., Xiong, G., Liu, H., Chen, F., Wang, L., Meng, X., Liu, G., Yu, H., Yuan, Y., Yi, W., Zhao, L., Ma, H., He, Y., Wu, Z., Melcher, K., Qian, Q., Xu, H.E., Wang, Y., and Li, J. (2013). DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 504, 401–405.

    CAS  Article  PubMed  Google Scholar 

  • Li, M., Li, X., Zhou, Z., Wu, P., Fang, M., Pan, X., Lin, Q., Luo, W., Wu, G., and Li, H. (2016). Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Front Plant Sci 7, 377.

    PubMed  PubMed Central  Google Scholar 

  • Li, T., Huang, S., Zhao, X., Wright, D.A., Carpenter, S., Spalding, M.H., Weeks, D.P., and Yang, B. (2011). Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 39, 6315–6325.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Liu, X., Zhang, Y., Cheng, C., Cheng, A.W., Zhang, X., Li, N., Xia, C., Wei, X., Liu, X., and Wang, H. (2017). CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res 27, 154–157.

    CAS  Article  PubMed  Google Scholar 

  • Ma, X., and Liu, Y.G. (2016). CRISPR/Cas9-based multiplex genome editing in monocot and dicot plants. Curr Protoc Mol Biol 115, 31.6.1–31.6.21.

    PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  • Ma, X., Chen, L., Zhu, Q., Chen, Y., and Liu, Y.G. (2015a). Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products. Mol Plant 8, 1285–1287.

    CAS  Article  PubMed  Google Scholar 

  • Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y., Xie, Y., Shen, R., Chen, S., Wang, Z., Chen, Y., Guo, J., Chen, L., Zhao, X., Dong, Z., and Liu, Y.G. (2015b). A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8, 1274–1284.

    CAS  Article  PubMed  Google Scholar 

  • Mao, H., Sun, S., Yao, J., Wang, C., Yu, S., Xu, C., Li, X., and Zhang, Q. (2010). Linking differential domain functions of the GS3 protein to natural variation of grain size in rice. Proc Natl Acad Sci USA 107, 19579–19584.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Mitsuda, S.H., and Shimizu, N. (2016). Epigenetic repeat-induced gene silencing in the chromosomal and extrachromosomal contexts in human cells. PLoS One 11, e0161288.

    Article  PubMed  PubMed Central  Google Scholar 

  • Miura, K., Ikeda, M., Matsubara, A., Song, X.J., Ito, M., Asano, K., Matsuoka, M., Kitano, H., and Ashikari, M. (2010). OsSPL14 promotes panicle branching and higher grain productivity in rice. Nat Genet 42, 545–549.

    CAS  Article  PubMed  Google Scholar 

  • Monna, L., Kitazawa, N., Yoshino, R., Suzuki, J., Masuda, H., Maehara, Y., Tanji, M., Sato, M., Nasu, S., and Minobe, Y. (2002). Positional cloning of rice semidwarfing gene, sd-1: rice “green revolution gene” encodes a mutant enzyme involved in gibberellin synthesis. DNA Res 9, 11–17.

    CAS  Article  PubMed  Google Scholar 

  • Patron, N.J. (2014). DNA assembly for plant biology: techniques and tools. Curr Opin Plant Biol 19, 14–19.

    CAS  Article  PubMed  Google Scholar 

  • Piao, R., Jiang, W., Ham, T.H., Choi, M.S., Qiao, Y., Chu, S.H., Park, J.H., Woo, M.O., Jin, Z., An, G., Lee, J., and Koh, H.J. (2009). Map-based cloning of the ERECT PANICLE 3 gene in rice. Theor Appl Genet 119, 1497–1506.

    CAS  Article  PubMed  Google Scholar 

  • Qi, W., Zhu, T., Tian, Z., Li, C., Zhang, W., and Song, R. (2016). High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNAprocessing system-based strategy in maize. BMC Biotechnol 16, 58.

    Article  PubMed  PubMed Central  Google Scholar 

  • Sakuma, T., Masaki, K., Abe-Chayama, H., Mochida, K., Yamamoto, T., and Chayama, K. (2016). Highly multiplexed CRISPR-Cas9-nuclease and Cas9-nickase vectors for inactivation of hepatitis B virus. Genes Cells 21, 1253–1262.

    CAS  Article  PubMed  Google Scholar 

  • Shan, Q., Wang, Y., Li, J., and Gao, C. (2014). Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9, 2395–2410.

    CAS  Article  PubMed  Google Scholar 

  • Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J.J., Qiu, J.L., and Gao, C. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31, 686–688.

    CAS  Article  PubMed  Google Scholar 

  • Shen, L., Wang, C., Fu, Y., Wang, J., Liu, Q., Zhang, X., Yan, C., Qian, Q., and Wang, K. (2016). QTL editing confers opposing yield performance in different rice varieties. J Integr Plant Biol in press, doi: 10.1111/jipb.12501.

    Google Scholar 

  • Si, L., Chen, J., Huang, X., Gong, H., Luo, J., Hou, Q., Zhou, T., Lu, T., Zhu, J., Shangguan, Y., Chen, E., Gong, C., Zhao, Q., Jing, Y., Zhao, Y., Li, Y., Cui, L., Fan, D., Lu, Y., Weng, Q., Wang, Y., Zhan, Q., Liu, K., Wei, X., An, K., An, G., and Han, B. (2016). OsSPL13 controls grain size in cultivated rice. Nat Genet 48, 447–456.

    CAS  Article  PubMed  Google Scholar 

  • Song, X.J., Huang, W., Shi, M., Zhu, M.Z., and Lin, H.X. (2007). A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nat Genet 39, 623–630.

    CAS  Article  PubMed  Google Scholar 

  • Sood, B.C., and Sidiq, E.A. (1978). A rapid technique for scent determination in rice. Indian J Genet Plant Breed 38, 268‒275.

    Google Scholar 

  • Spielmeyer, W., Ellis, M.H., and Chandler, P.M. (2002). Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene. Proc Natl Acad Sci USA 99, 9043–9048.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Wang, C., Shen, L., Fu, Y., Yan, C., and Wang, K. (2015a). A Simple CRISPR/Cas9 system for multiplex genome editing in rice. J Genets Genomics 42, 703–706.

    Article  Google Scholar 

  • Wang, S., Li, S., Liu, Q., Wu, K., Zhang, J., Wang, S., Wang, Y., Chen, X., Zhang, Y., Gao, C., Wang, F., Huang, H., and Fu, X. (2015b). The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nat Genet 47, 949–954.

    CAS  Article  PubMed  Google Scholar 

  • Wang, X., Niu, Y., Zhou, J., Yu, H., Kou, Q., Lei, A., Zhao, X., Yan, H., Cai, B., Shen, Q., Zhou, S., Zhu, H., Zhou, G., Niu, W., Hua, J., Jiang, Y., Huang, X., Ma, B., and Chen, Y. (2016). Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep. Sci Rep 6, 32271.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Wood, A.J., Lo, T.W., Zeitler, B., Pickle, C.S., Ralston, E.J., Lee, A.H., Amora, R., Miller, J.C., Leung, E., Meng, X., Zhang, L., Rebar, E.J., Gregory, P.D., Urnov, F.D., and Meyer, B.J. (2011). Targeted genome editing across species using ZFNs and TALENs. Science 333, 307–307.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Wu, X., Tang, D., Li, M., Wang, K., and Cheng, Z. (2013). Loose plant architecture1, an INDETERMINATE DOMAIN protein involved in shoot gravitropism, regulates plant architecture in rice. Plant Physiol 161, 317–329.

    CAS  Article  PubMed  Google Scholar 

  • Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14, 327.

    Article  PubMed  PubMed Central  Google Scholar 

  • Yano, M., Katayose, Y., Ashikari, M., Yamanouchi, U., Monna, L., Fuse, T., Baba, T., Yamamoto, K., Umehara, Y., Nagamura, Y., and Sasaki, T. (2000). Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the arabidopsis flowering time gene CONSTANS. Plant Cell 12, 2473–2484.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Yu, H., Murchie, E.H., González-Carranza, Z.H., Pyke, K.A., and Roberts, J.A. (2015). Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development. J Exp Bot 66, 1543–1552.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Zhang, Z., Mao, Y., Ha, S., Liu, W., Botella, J.R., and Zhu, J.K. (2016). A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Rep 35, 1519–1533.

    CAS  Article  PubMed  Google Scholar 

  • Zhou, Y., Zhu, J., Li, Z., Yi, C., Liu, J., Zhang, H., Tang, S., Gu, M., and Liang, G. (2009). Deletion in a quantitative trait gene qPE9-1 associated with panicle erectness improves plant architecture during rice domestication. Genets 183, 315–324.

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31271681, 3140101312), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and Jiangsu Agriculture Science and Technology Innovation Fund (CX(13)5075).

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Affiliations

  1. State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310006, China

    Lan Shen, Yufeng Hua, Yaping Fu, Qing Liu, Xiaozhen Jiao, Junjie Wang & Kejian Wang

  2. Key Laboratory of Plant Functional Genomics, Ministry of Education, Yangzhou University, Yangzhou, 225009, China

    Lan Shen & Changjie Yan

  3. Lianyungang Academy of Agricultural Sciences, Lianyungang, 222006, China

    Jian Li

  4. College of Life Sciences, Shanxi Agricultural University, Taigu, 030801, China

    Gaowei Xin & Xingchun Wang

Authors
  1. Lan Shen
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  2. Yufeng Hua
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  3. Yaping Fu
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  4. Jian Li
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  5. Qing Liu
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  6. Xiaozhen Jiao
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  7. Gaowei Xin
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  8. Junjie Wang
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  9. Xingchun Wang
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  10. Changjie Yan
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  11. Kejian Wang
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Correspondence to Changjie Yan or Kejian Wang.

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Shen, L., Hua, Y., Fu, Y. et al. Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice. Sci. China Life Sci. 60, 506–515 (2017). https://doi.org/10.1007/s11427-017-9008-8

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  • DOI: https://doi.org/10.1007/s11427-017-9008-8

Keywords

  • CRISPR/Cas9
  • genome editing
  • agronomic genes
  • rice