Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice

Rice - 2014
Ruihua Xu1, Hao Li1, Ruiying Qin1, Lu Wang1, L i Li1, Pengcheng Wei2, Jian‐Bo Yang1
1Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, 230031, China
2Institute of Agricultural Engineering, Anhui Academy of Agricultural Sciences, Hefei, 230031, China

Tóm tắt

Abstract Background The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation. Findings Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon. Conclusions Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.

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Tài liệu tham khảo

Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V: Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 2013, 9(1):39. 10.1186/1746-4811-9-39

Chen K, Gao C: TALENs: customizable molecular DNA scissors for genome engineering of plants. J Genet Genomics 2013, 40(6):271–279. [http://dx.doi.org/10.1016/j.jgg.2013.03.009] 10.1016/j.jgg.2013.03.009

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F: Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339(6121):819–823. 10.1126/science.1231143

Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy R, Fauquet C: Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 2001, 7(1):25–33. 10.1023/A:1009687511633

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, ᅟ: ᅟ.

Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O’Connor-Giles KM: Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 2013, ᅟ: ᅟ.

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F: DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotech 2013, 31(9):827–832. [http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html#supplementary-information] http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html#supplementary-information http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html#supplementary-information 10.1038/nbt.2647

Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP: Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 2013, 41(20):e188. 10.1093/nar/gkt780

Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M: Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotech 2013, 31(8):681–683. [http://www.nature.com/nbt/journal/v31/n8/full/nbt.2661.html#supplementary-information] http://www.nature.com/nbt/journal/v31/n8/full/nbt.2661.html#supplementary-information http://www.nature.com/nbt/journal/v31/n8/full/nbt.2661.html#supplementary-information 10.1038/nbt.2661

Li J-F, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J: Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotech 2013, 31(8):688–691. [http://www.nature.com/nbt/journal/v31/n8/full/nbt.2654.html#supplementary-information] http://www.nature.com/nbt/journal/v31/n8/full/nbt.2654.html#supplementary-information http://www.nature.com/nbt/journal/v31/n8/full/nbt.2654.html#supplementary-information 10.1038/nbt.2654

Li T, Liu B, Spalding MH, Weeks DP, Yang B: High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotech 2012, 30(5):390–392. [http://www.nature.com/nbt/journal/v30/n5/abs/nbt.2199.html#supplementary-information] http://www.nature.com/nbt/journal/v30/n5/abs/nbt.2199.html#supplementary-information http://www.nature.com/nbt/journal/v30/n5/abs/nbt.2199.html#supplementary-information 10.1038/nbt.2199

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM: RNA-guided human genome engineering via Cas9. Science 2013, 339(6121):823–826. 10.1126/science.1232033

Mao Y, Zhang H, Xu N, Zhang B, Gao F, J-k Z: Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 2013, ᅟ: ᅟ.

Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu L-J: Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 2013, 23(10):1233–1236. 10.1038/cr.2013.123

Nekrasov V, Staskawicz B, Weigel D, Jones JDG, Kamoun S: Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotech 2013, 31(8):691–693. [http://www.nature.com/nbt/journal/v31/n8/full/nbt.2655.html#supplementary-information] http://www.nature.com/nbt/journal/v31/n8/full/nbt.2655.html#supplementary-information http://www.nature.com/nbt/journal/v31/n8/full/nbt.2655.html#supplementary-information 10.1038/nbt.2655

Pan G, Zhang X, Liu K, Zhang J, Wu X, Zhu J, Tu J: Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides. Plant Mol Biol 2006, 61(6):933–943. 10.1007/s11103-006-0058-z

Shan Q, Wang Y, Chen K, Liang Z, Li J, Zhang Y, Zhang K, Liu J, Voytas DF, Zheng X, Zhang Y, Gao C: Rapid and efficient gene modification in rice and brachypodium using TALENs. Mol Plant 2013, ᅟ: ᅟ.

Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu J-L, Gao C: Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotech 2013, 31(8):686–688. [http://www.nature.com/nbt/journal/v31/n8/full/nbt.2650.html#supplementary-information] http://www.nature.com/nbt/journal/v31/n8/full/nbt.2650.html#supplementary-information http://www.nature.com/nbt/journal/v31/n8/full/nbt.2650.html#supplementary-information 10.1038/nbt.2650

Wang H, Yang H, Shivalila Chikdu S, Dawlaty Meelad M, Cheng Albert W, Zhang F, Jaenisch R: One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013, 153(4):910–918. 10.1016/j.cell.2013.04.025

Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, Zhu Z, Lin S, Zhang B: Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 2013, ᅟ: ᅟ.

Xie K, Yang Y: RNA-guided genome editing in plants using a CRISPR-cas system. Mol Plant 2013, ᅟ: ᅟ.

Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF: Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 2013, 161(1):20–27. 10.1104/pp.112.205179

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