Chinese Journal OF Rice Science >
Orientation Improvement of Grain Length and Grain Number in Rice by Using CRISPR/Cas9 System
Received date: 2017-03-09
Revised date: 2017-03-20
Online published: 2017-05-10
【Objective】Gene orientation editing has become an important way for molecular breeding. We evaluated the improvement effects on the target traits following the construction of GS3 and Gn1a loss-of-function mutants so as to lay a solid foundation for high-yielding rice breeding. 【Method】GS3 and Gn1a were selected as targets for gene editing, which control the grain size and grain number in rice, respectively. The pC1300-2×35S:: Cas9-gGS3-gGn1a expression vector was constructed for knocking out both GS3 and Gn1a by using CRISPR/Cas9 system, and transformed into four good quality rice varieties by the Agrobacterium-mediated method. And the properties of mutations and the target traits were analyzed in the transgenic lines.【Result】The sequencing results showed the GS3 and Gn1a in four rice varieties were successfully edited. In T0 generation, we obtained mutants with frame shift mutations in GS3 and Gn1a in four rice varieties. In T1 generation, the marker-free plants were screened for analyzing the agronomic traits in four genetic backgrounds. For the agronomic characters, the gs3 and gs3gn1a mutants had the longer grain length and the increased 1000-grain weight compared to the wild type, and the gs3gn1a mutants had more grains per panicle compared to the gs3 mutants.【Conclusion】CRISPR/Cas9-mediated gene editing can improve rice target traits, which has great potential in orientation improvement of rice varieties.
Key words： CRISPR/Cas9; gene editing; rice; GS3; Gn1a
Lan SHEN, Jian LI, Yaping FU, Junjie WANG, Yufeng HUA, Xiaozhen JIAO, Changjie YAN, Kejian WANG . Orientation Improvement of Grain Length and Grain Number in Rice by Using CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2017 , 31(3) : 223 -231 . DOI: 10.16819/j.1001-7216.2017.7029 223
| [1] | Wiedenheft B, Sternberg S H, Doudna J A.RNA-guided genetic silencing systems in bacteria and archaea.Nature, 2012, 482: 331-338. |
| [2] | Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F.Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339: 819-823. |
| [3] | Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J L, Gao C.Targeted genome modification of crop plants using a CRISPR/Cas system. Nat Biotechnol, 2013, 31: 686-688. |
| [4] | DiCarlo J E, Norville J E, Mali P, Rios X, Aach J, Church G M. Genome engineering in Saccharomyces cerevisiae using CRISPR/Cas systems.Nucleic Acids Res, 2013, 41: 4336-4343. |
| [5] | Jakociunas T, Bonde I, Herrgard M, Harrison SJ, Kristensen M, Pedersen L E, Jensen M K, Keasling J D.Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae.Metab Eng, 2015, 28: 213-222. |
| [6] | Zhou X, Xin J, Fan N, Zou Q, Huang J, Ouyang Z, Zhao Y, Zhao B, Liu Z, Lai S, Yi X, Guo L, Esteban M A, Zeng Y, Yang H, Lai L.Generation of CRISPR/Cas 9-mediated gene-targeted pigs via somatic cell nuclear transfer.Cell Mol Life Sci, 2015, 72: 1175-1184. |
| [7] | Zou Q, Wang X, Liu Y, Ouyang Z, Long H, Wei S, Xin J, Zhao B, Lai S, Shen J, Ni Q, Yang H, Zhong H, Li L, Hu M, Zhang Q, Zhou Z, He J, Yan Q, Fan N, Zhao Y, Liu Z, Guo L, Huang J, Zhang G, Ying J, Lai L, Gao X.Generation of gene-target dogs using CRISPR/Cas 9 system.J Mol Cell Biol, 2015, 7: 580-583. |
| [8] | Wang H, Yang H, Shivalila C S, Dawlaty M M, Cheng A W, Zhang F, Jaenisch R.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas- mediated genome engineering.Cell, 2013, 153: 910-918. |
| [9] | Hoshijima K, Jurynec M J, Grunwald D J.Precise editing of the zebrafish genome made simple and efficient.Dev Cell, 2016, 36: 654-667. |
| [10] | Gratz S J, Harrison M M, Wildonger J, O'Connor-Giles K M. Precise genome editing of drosophila with CRISPR/Cas RNA-guided Cas9.Methods Mol Biol, 2015, 1311: 335-348. |
| [11] | Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant, 2013, 6: 2008-2011. |
| [12] | Puchta H.Applying crispr/cas for genome engineering in plants: The best is yet to come.Curr Opin Plant Biol, 2016, 36: 1-8. |
| [13] | Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks D P.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.Nucleic Acids Res, 2013, 41: e188. |
| [14] | Feng Z, Zhang B, Ding W, Liu X, Yang D L, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu J K.Efficient genome editing in plants using a CRISPR/Cas system.Cell Res, 2013, 23: 1229-1232. |
| [15] | Wang C, Shen L, Fu Y, Yan C, Wang K.A simple crispr/cas9 system for multiplex genome editing in rice. J Genet Genom, 2015, 42: 703-706. |
| [16] | Zhang Z J, Mao Y F, Ha S, Liu W S, Botella J R, Zhu J K.A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis.Plant Cell Rep, 2016, 35: 1519-1533. |
| [17] | Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol Plant, 2015, 8: 1274-1284. |
| [18] | Ma X, Chen L, Zhu Q, Chen Y, Liu Y G.Rapid decoding of sequence-specific nuclease-induced heterozygous and biallelic mutations by direct sequencing of PCR products.Mol Plant, 2015, 8: 1285-1287. |
| [19] | Xu R F, Li H, Qin R Y, Li J, Qiu C H, Yang Y C, Ma H, Li L, Wei P C, Yang J B.Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system.Sci Rep, 2015, 5: 11491 |
| [20] | Li J, Sun Y, Du J, Zhao Y, Xia L.Generation of targeted point mutations in rice by a modified CRISPR/CAS9 system. Mol Plant, 2017, 10(3): 526-529. |
| [21] | Lu Y, Zhu J K.Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system.Mol Plant, 2017, 10(3): 523-525. |
| [22] | Fan C, Xing Y, Mao H, Lu T, Han B, Xu C, Li X, Zhang Q.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, 2006, 112: 1164-1171. |
| [23] | Mao H, Sun S, Yao J, Wang C, Yu S, Xu C, Li X, Zhang Q.Linking differential domain functions of theGS3 protein to natural variation of grain size in rice.Proc Natl Acad Sci U S A, 2010, 107: 19579-19584. |
| [24] | Ashikari M, Sakakibara H, Lin S, Yamamoto T, Takashi T, Nishimura A, Angeles E R, Qian Q, Kitano H, Matsuoka M.Cytokinin oxidase regulates rice grain production.Science, 2005, 309: 741-745. |
| [25] | Shen L, Wang C, Fu Y, Wang J, Liu Q, Zhang X, Yan C, Qian Q, Wang K.QTL editing confers opposing yield performance in different rice varieties.J Integr Plant Biol, 2016, DOI: 10.1111/jipb.12501 |
| [26] | Hiei Y, Ohta S, Komari T, Kumashiro T.Efficient transformation of rice(Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the t-DNA.Plant J, 1994, 6: 271-282. |
| [27] | Li M, Li X, Zhou Z, Wu P, Fang M, Pan X, Lin Q, Luo W, Wu G, Li H. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Front Plant Sci, 2016, 7: |
| [28] | Feng C, Yuan J, Wang R, Liu Y, Birchler J A, Han F.Efficient targeted genome modification in maize using CRISPR/Cas 9 system. J Genet Genom, 2016, 43: 37-43. |
| [29] | Svitashev S, Young J K, Schwartz C, Gao H, Falco S C, Cigan A M.Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA.Plant Physiol, 2015, 169: 931-945 |
| [30] | Shan Q, Wang Y, Li J, Gao C.Genome editing in rice and wheat using the CRISPR/Cas system.Nat Protoc, 2014, 9: 2395-2410. |
| [31] | Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol, 2014, 32: 947-951. |
| [32] | Lawrenson T, Shorinola O, Stacey N, Li C, Ostergaard L, Patron N, Uauy C, Harwood W.Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guidedCas9 nuclease. Genome Biol, 2015, 16: 258. |
| [33] | Cai Y, Chen L, Liu X, Sun S, Wu C, Jiang B, Han T, Hou W.CRISPR/Cas 9-mediated genome editing in soybean hairy roots.PLoS One, 2015, 10: e0136064. |
| [34] | Jacobs T B, LaFayette P R, Schmitz R J, Parrott W A. Targeted genome modifications in soybean with CRISPR/Cas 9.BMC Biotechnol, 2015, 15: 16. |
| [35] | Xing Y, Zhang Q.Genetic and molecular bases of rice yield.Annu Rev Plant Biol, 2010, 61: 421-442. |
/
| 〈 |
|
〉 |