DOI: 10.3724/SP.J.1006.2018.00227

Acta Agronomica Sinica (作物学报) 2018/44:2 PP.227-235

Establishment of CRISPR/Cas9 Genome Editing System Based on GbU6 Promoters in Cotton (Gossypium barbadense L.)

CRISPR/Cas9 genome editing is a powerful tool for genes functional analyses, and the mutation of endogenous genes has been successfully implemented in many organisms using the tool. Two cloned U6 promoter from sea island cotton Xinhai 16 were used to construct CRISPR/Cas9 gene editing vectors with target (GbGGB and GBERA1) DNA fragments from Xinhai 16 respectively. Through PEG method, the core fragments (including GbU6::sgRNA and CAMV35S::Cas9) of the CRISPR/Cas9 gene editing vectors enriched by PCR method were transformed into the cotton protoplast prepared from the embryo callus of Xinhai 16. The mutation of endogenous target genes was successfully detected by a restriction enzyme PCR (RE-PCR) assay of protoplast genome. The cloning and sequencing of the PCR product, showed that the two Cas9-GbU6-sgRNA vectors could both induce targeted mutagenesis. Sequence analysis revealed that most of the mutations were nucleotide substitutions and the few were nucleotide deletion. The results indicate that the CRISPR/Cas9 gene editing vector system based on GbU6 promoter can realize targeted mutagenesis in sea island cotton, which provides an important technical basis for cotton functional genomics research.

Key words:cotton,protoplast,CRISPR/Cas9,genome editing

ReleaseDate:2018-03-08 10:13:39

[1] 李付广, 袁有禄, 棉花分子育种学. 北京:中国农业大学出版社, 2013 Li F G, Yuan Y L. Molecular Breeding of Cotton. Beijing:China Agricultural University Press, 2013(in Chinese)

[2] 张德超. 棉花叶片干旱胁迫蛋白的表达分析与鉴定. 中国农业科学院硕士学位论文, 北京, 2013 Zhang D C. Analysis and Identification of Drought Stress Proteins in Cotton Leaves. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing, China, 2013(in Chinese)

[3] 丁震乾, 陈天子, 刘廷利, 刘小双, 张保龙, 周兴根. 棉花干旱诱导MYB类转录因子GhRAX3的功能分析. 中国农业科学, 2015, 18:3569-3579 Ding Z Q, Chen T Z, Liu T L, Liu X S, Zhang B L, Zhou G X. Functional analysis of MYB transcription factor GhRAX3 induced by Cotton Drought. Sci Agric Sin, 2015, 18:3569-3579(in Chinese with English abstract)

[4] 许宗弘. 棉花枯黄萎病研究现状及展望. 知识经济, 2010, (16):132 Xu Z H. Research status and Prospect of cotton wilt disease. Knowledge Economy, 2010, (16):132(in Chinese)

[5] 任爱霞. 棉花枯黄萎病抗性遗传及生化机理研究. 浙江大学硕士学位论文, 浙江杭州, 2002 Ren A X. Genetic and Biochemical Mechanism of Resistance to Verticillium wilt of Cotton. MS Thesis of Zhejiang University, Hangzhou, China, 2002(in Chinese)

[6] 戴敬, 徐俊兵, 杨举善, 吕丽兰. 棉花留叶枝栽培的研究现状与应用前景. 中国农业科技导报, 2003, (6):19-23 Dai J, Xu J B, Yang J H, Lyu L L. Study on the cultivation of cotton retaining with status and application prospect. J Agric Sci Technol, 2003, (6):19-23(in Chinese with English abstract)

[7] 徐立华. 我国棉花高产、高效栽培技术研究现状与发展思路. 中国棉花, 2001, (3):5-8 Xu L H. Research status and development of high yield and high efficiency cultivation techniques of cotton in China. China Cotton, 2001, (3):5-8(in Chinese)

[8] 孙学振, 施培, 周治国. 我国棉花高产栽培技术理论研究现状与展望. 中国棉花, 1999, (4):2-7 Sun X Z, Shi P, Zhou Z G. Current situation and prospect of high yield cultivation techniques of cotton in China. China Cotton, 1999, (4):2-7(in Chinese)

[9] Sun Y, Li J, Xia L. Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement. Front Plant Sci, 2016, 7:1928

[10] Cao H X, Wang W, Le H T T, Giang T H Vu. The power of CRISPR-Cas9-induced genome editing to speed up plant breeding. Int J Genom, 2016:5078796

[11] Gilbert L A, Larson H M, Morsut L, Z R Liu, Brar G A, Torres S E, Ginossar N S, Brandman O, Whitehead E H, Doudna J A, Lim W A, Weissman J S, Qi L S. CRISPR-mediated modular RNA-guided regulation of transcription in Eukaryotes. Cell, 2013, 154:442-451

[12] Hsu P D, Lander E S, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157:1262-1278

[13] Bassett A R, C Tibbit, Ponting C P, Liu J L. Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system. Cell Rep, 2013, 4:220-228

[14] Barrangou R, Marraffini L A. CRISPR-Cas systems:prokaryotes upgrade to adaptive immunity. Mol Cell, 2014, 54:234-244

[15] Mao Y F, Zhang Z G, Feng Z Y, Wei P L, Zhang H, Botella J R, Zhu J K. Development of germline specific CRISPR/Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol J, 2016, 14:519-532

[16] Kim H, Kim S T, Ryu J, Choi M K, Kweon J, Kang B C, Ahn S M, Bae S J, Kim J G, Kim J S, Kim S G. A simple flexible and high-throughput cloning system for plant genome editing via CRISPR/Cas system. J Integr Plant Biol, 2016, 58:705-712

[17] Gao S L, Tong Y Y, Wen Z Q. Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR/Cas9 system. Microbiol Biotech, 2016, 43:1085-1093

[18] Johnson R A, Gurevich V, Filler S, Samach A, Levy A A. Comparative assessments of CRISPR-Cas nucleases cleavage efficiency in planta. Plant Mol Biol, 2015, 87:143-156

[19] Xu R F, Li H, Qin R Y, Wang L, Li L, Wei P C, Yang J B. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice, 2014, 7:5

[20] Kumar V, Jain M. The CRISPR/Cas system for plant genome editing:advances and opportunities. J Exp Bot, 2015, 66:47-57

[21] Sharma K, Hrle A, Kramer K, Sachsenberg T, Staals R H J, Randau L, Marchfelder A, van der Oost J, Kohlbacher O, Conti E, Urlaub H. Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry. Methods, 2015, 89:138-148

[22] Basak J, Nithin C. Targeting non-coding RNAs in plants with the CRISPR-Cas technology is a challenge yet worth accepting. Front Plant Sci, doi:10.3389/fpls.2015.01001

[23] Zlotorynski E. Plant cell biology:CRISP-Cas protection from plant viruses. Nat Rev Mol Cell Biol, 2015, 16:642

[24] Liu L, Fan X D. CRISPR-Cas system:a powerful tool for genome engineering. Plant Mol Biol, 2014, 3:209-218

[25] Chen X, Lu X, Shu N, Wang S, Wang J, Wang D, Guo L, Ye W W. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system. Sci Rep, 2017, 7:44304

[26] Li C, Unver T, Zhang B. A high efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton (Gossypium hirsutum L.). Sci Rep, 2017, 7:43902

[27] Johnson C D, Chary S N, Chernoff E A, Zeng Q, Running M P, Crowell D N. Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis. Plant Physiol, 2005, 139:722-733

[28] Andrews M, Huizinga D H, Crowell D N. The CaaX specificities of Arabidopsis proteinprenyltransferases explain era1 and ggb phenotypes. BMC Plant Biol, 2010, 10:118

[29] 雷建峰, 徐新霞, 李月, 代培红, 刘超, 刘晓东. CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定. 西北植物学报, 2016, 36:857-864 Lei J F, Xu X X, Li Y, Dai P H, Liu C, Liu X D. CRISPR/Cas9 mediated targeting to in identification of GGB mutants of Arabidopsis. J Northwest Plants, 2016, 36:857-864

[30] 雷建峰, 伍娟, 陈晓俊, 於添平, 倪志勇, 李月, 张巨松, 刘晓东. 棉花花粉中高效转录U6启动子的克隆及功能分析. 中国农业科学, 2015, 48:3794-3802 Lei J F, Wu J, Chen X J, Yu T P, Ni Z Y, Li Y, Zhang J S, Liu X D. cloning and functional analysis of high efficient U6 promoter in cotton pollen. Chin J Agric Sci, 2015, 48:3794-3802

[31] Lu Y M, Chen X, Wu Y X, Wang Y P, He Y Q, Wu Y. Directly transforming PCR amplified DNA fragments into plant cells is a versatile system That facilitates the transient expression assay. PLoS One, 2013, 8:e57171