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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Patron, N.J. (2014). DNA assembly for plant biology: techniques and tools. Curr Opin Plant Biol 19, 14–19.
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.
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.
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.
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.
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.
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.
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.
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.
Sood, B.C., and Sidiq, E.A. (1978). A rapid technique for scent determination in rice. Indian J Genet Plant Breed 38, 268‒275.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Contributed equally to this work
Electronic supplementary material
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-017-9008-8