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作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (11): 2706-2714.doi: 10.3724/SP.J.1006.2022.14220

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆多基因编辑表达载体的构建及应用

陈向前(), 姜奇彦(), 孙现军, 牛风娟, 张慧媛, 胡正*(), 张辉*()   

  1. 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2021-11-26 接受日期:2022-02-25 出版日期:2022-11-12 网络出版日期:2022-03-08
  • 通讯作者: 胡正,张辉
  • 作者简介:第一作者联系方式: 陈向前, E-mail: chen810702351@gmail.com;
    姜奇彦, E-mail: jiangqiyan@caas.cn第一联系人：** 同等贡献。
  • 基金资助:
    本研究由海南省重点研发计划项目(ZDYF2022XDNY135);中国农业科学院科技创新工程(CAAS-ZDRW202201);国家重点研发计划项目(2021YFD1201603-2);国家自然科学基金项目(31601302)

Construction and application of soybean CRISPR/Cas9 multiplex editing vector

CHEN Xiang-Qian(), JIANG Qi-Yan(), SUN Xian-Jun, NIU Feng-Juan, ZHANG Hui-Yuan, HU Zheng*(), ZHANG Hui*()   

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2021-11-26 Accepted:2022-02-25 Published:2022-11-12 Published online:2022-03-08
  • Contact: HU Zheng,ZHANG Hui
  • Supported by:
    The Key Research and Development Program of Hainan Province(ZDYF2022XDNY135);The Agricultural Science and Technology Program for Innovation Program(CAAS-ZDRW202201);The National Key Research and Development Program of China(2021YFD1201603-2);The National Natural Science Foundation of China(31601302)

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摘要：

大豆基因家族往往存在多个功能相似的基因, 开发多基因编辑载体对多基因或基因家族进行编辑, 对遗传转化效率低的大豆的基因编辑及基因功能研究具有重要的应用价值。本研究利用大豆特有的不同U6启动子驱动表达sgRNA, 利用载体上同尾酶将不同大豆U6启动子表达的sgRNA进行串联, 成功构建了可以同时对大豆多个基因进行编辑的CRISPR/Cas9多基因编辑表达载体pCambia3301-Cas9-GmU6n-gDNAn。并利用该载体, 在大豆发状根中成功实现了大豆GRF (Growth-Regulating Factor)基因家族不同成员同时被编辑的目的。该载体的创建有效提高了大豆基因编辑效率, 为大豆基因编辑及基因功能研究提供重要的工具。

关键词: 大豆, U6启动子, CRISPR/Cas9多基因编辑载体, 发状根

Abstract:

Most members of one gene family have similar function in soybean. Construction of soybean multiplex editing vector to edit multiple genes or gene families have important application value in soybean gene editing and gene functions, especially for the soybean with low transformation efficiency. Here, we reported a CRISPR/Cas9 multiplex editing vector, pCambia3301- Cas9-GmU6n-gDNAn, that enabled the editing of multiple genes in soybean. In this vector, different sgRNAs were driven by different soybean U6 promoters, and multiple sgRNA expression cassettes were assembled into pCambia3301-Cas9 vector by isocaudamers. The result showed that this system could simultaneously produce multiple mutations in soybean hairy roots by targeting multiple GRF genes via single transformation events. This vector can improve the efficiency of gene editing in soybean, and provide a simple toolbox for studying functions of multiple genes and gene families in soybean for basic research and genetic improvement.

Key words: soybean, GmU6 promoter, CRISPR/Cas9 multiplex editing vector, hairy root

表1

本研究所用引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
载体构建引物Primers used in construction of vectors
PDS-HF GAAATGTGCCACCACATGGATTggtgaccGCGCACAACCCTTTCCAGTTTCT
PDS-HR AACTTGCTATTTCTAGCTCTAAAAcctaggCATCATAGTTACTAACCTCACT
GmU6-2-HF TGTAAAACGACGGCCAGAGAATTCGAGCTCGAGTCCAATATGCCC
GmU6-2-HR AACTGGAAAGGGTTGTGCGCGGTCACCAACATCTTGAATGTTGTATGTC
GmU6-8-HF TGTAAAACGACGGCCAGAGAATTCGAGCTCGAGATTCGAGCTCGA
GmU6-8-HR AACTGGAAAGGGTTGTGCGCGGTCACCAATCCATATGTTTTCCTGGGACT
GmU6-11-HF TGTAAAACGACGGCCAGAGAATTCGAGCTCGAGATGGTCTATGAG
GmU6-11-HR AACTGGAAAGGGTTGTGCGCGGTCACCAACCAGTTTGTTCCATCTCTG
串联sgRNA构建引物Primers used in assembling of multiple sgRNA expression cassettes
U2-GRF TGACATACAACATTCAAGATGTTGAAACCGTTCAAGAAAGCCTGGTTTTAGAGCTAGAAATAGCAAGT
U8-GRF TAGTCCCAGGAAAACATATGGATTGCCACAGGCTTTCTTGAACGAGTTTTAGAGCTAGAAATAGCAAGT
U10-GRF GAAATGTGCCACCACATGGATTGGTTCCACAGGCTTTCTTGAAGTTTTAGAGCTAGAAATAGCAAGT
U11-GRF CAGAGATGGAACAAACTGGTTGGAATCGTTCAAGAAAGCCTGGTTTTAGAGCTAGAAATAGCAAGT
GRF突变检测PCR引物Primers used in the detection of GRF mutations
GRF5-F/R GAGCCAGGGAGGTGTAGGAG/AATGTGATGAATGGAATAAACGA
GRF6-F/R ATGGAAAGAAATGGCGATGC/ACCAACCACAAACGAGGAAA
GRF7-F/R ATGGGATGAGGATGATGATGT/GAGGAATGAGGAGGAATAGGG
GRF8-F/R GAGCCAGGCAGGTGTAGGAG/ACGAATGAGGTGGAAGAGGG
GRF9-F/R CACATCCTCTTCTCGCTCTTC/GAAACTCACCTGGGTTCCTTAT
GRF10-F/R GTTCACAGTGGATGGTGGTTT/CAAGGTATGGCTGAGTGGTAGTA
GRF13-F/R GCAGGAGGACTGATGGAAAA/TACTTGGAATCGCATAGGGA
GRF14-F/R CCATTTTGTCTTTATTGCTGC/TGGGAAGGTTTTGGAAGTTTA
GRF15-F/R TGTTGATTGGTTATTTGGTGC/TGTGATAGTGGGTTTGGTCTGA
GRF16-F/R ATATTAGTGATAAAGTAGAGGTGGGA/AACTCAATAACAAGCAGACGC
GRF17-F/R ATGAGCGTTCCTCCGCCGTCT/CCCTTGCGCCATTGATGTGC
GRF18-F/R TGACCTCCTTCTCCCCATTC/CAGTTGCAGTTCCAGAAACATTA
GRF19-F/R AATCAGGGTATTGGGGTAGAG/AGGGTGGTGAACAAATGGAA
GRF20-F/R AGGGTATTGGGGTAGAGGAGC/CGAAGAAGATCAAATGGGGAT
GRF21-F/R TCAGGGTACTACTGGCGAAGA/TGACCGAGGTGAAGGAGATT

图1

不同大豆U6启动子驱动的sgRNA3.0载体的构建 A和B: pGmU6-sgRNA3.0表达载体构建, M: DL2000; A1: PDS PCR扩增; A2: pGmU6-10-sgRNA2.0 Bsa I酶切; A3: pGmU6-10-sgRNA3.0 BstE II和Avr II双酶切。B1: pGmU6-2-sgRNA3.0 BstE II和Xho I双酶切, B2: GmU6-2, B3: GmU6-8, B4: GmU6-11。C: pGmU6-sgRNA3.0表达载体图。"

图2

pCambia3301-Cas9-GmU6n-gDNAn载体构建 A: pGmU6-sgRNA3.0载体BstE II和Avr II双酶切。M: DL2000; 1~4: pGmU6-2/8/10/11-sgRNA3.0载体酶切。B: pGmU6-gDNA载体双酶切。M: DL2000; 1: pGmU6-10-gDNA BamH I和Sal I双酶切; 2~4: pGmU6-2-gDNA、pGmU6-8-gDNA、pGmU6-11-gDNA载体分别用BamH I和Xho I双酶切。C: pCambia3301-Cas9-GmU6n-gDNAn载体构建。M: 1 kb ladder; 1: pCambia3301-Cas9载体用EcoR I和Hind III双酶切; 2: pGmU6n-gDNAn载体用EcoR I和Hind III双酶切; 3: pCambia3301-Cas9-GmU6n-gDNAn载体; 4: pCambia3301-Cas9-GmU6n-gDNAn载体用EcoR I和Hind III双酶切。"

图3

多靶位点Cas9载体示意图 Cas9和NLS融合后由35S启动表达。sgRNA由不同的大豆U6启动子启动表达。合成的多基因编辑载体骨架由串联排列的GmU6-gDNA-sgRNA单元组成, 每个单元包含一个大豆U6启动子, 一个识别具体基因的gDNA序列和保守的sgRNA结构。LB: 左边界; RB: 右边界。"

图4

PCR-T7 Endonuclease I酶切法检测GRF基因突变 图中1~12泳道代表不同单根DNA中扩增的GRF基因的酶切产物, 没有突变目的条带单一, 有突变的目的条带不单一(箭头标识)。M: 分子量marker, DL2000。"

图5

同一条转基因发状根中扩增GRF基因并测序检测突变 野生型靶基因序列中PAM位点用红色字母标识, gDNA用蓝色标识。GRFn表示不同GRF基因的突变序列, 虚线表示缺失碱基, 括号内数据表示缺失突变碱基数量。"

[1] Wyman C, Kanaar R. DNA double-strand break repair: all’s well that ends well. Annu Rev Genet, 2006, 40: 363-383.
doi: 10.1146/annurev.genet.40.110405.090451
[2] Li J F, Norville J E, Aach J, McCormack M, Zhang D, Bush J, Church G M, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol, 2013, 31: 688-691.
doi: 10.1038/nbt.2654
[3] Li W, Teng F, Li T, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol, 2013, 31: 684-686.
doi: 10.1038/nbt.2652
[4] 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.
doi: 10.1038/nbt.2650
[5] Zhan X, Lu Y, Zhu J K, Botella J R. Genome editing for plant research and crop improvement. J Integr Plant Biol, 2021, 63: 3-33.
doi: 10.1111/jipb.13063
[6] Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D L, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu J K. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA, 2014, 111: 4632-4637.
doi: 10.1073/pnas.1400822111
[7] Jiang W Z, Yang B, Weeks D P. Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations. PLoS One, 2014, 9: e99225.
doi: 10.1371/journal.pone.0099225
[8] Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z, Mao Y, Yang L, Zhang H, Xu N, Zhu J K. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J, 2014, 12: 797-807.
doi: 10.1111/pbi.12200 pmid: 24854982
[9] 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.
doi: 10.1038/nbt.2969
[10] Zheng M, Zhang L, Tang M, Liu J, Liu H, Yang H, Fan S, Terzaghi W, Wang H, Hua W. Knockout of two bnamax1 homologs by CRIPSR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.). Plant Biotechnol J, 2020, 18: 644-654.
doi: 10.1111/pbi.13228 pmid: 31373135
[11] Li B, Liang S, Alariqi M, Wang F, Wang G, Wang Q, Xu Z, Yu L, Naeem Zafar M, Sun L, Si H, Yuan D, Guo W, Wang Y, Lindsey K, Zhang X, Jin S. The application of temperature sensitivity CRISPR/LbCpf1 (LbCas12a) mediated genome editing in allotetraploid cotton (G. hirsutum) and creation of nontransgenic, gossypol-free cotton. Plant Biotechnol J, 2021, 19: 221-223.
doi: 10.1111/pbi.13470
[12] Pramanik D, Shelake R M, Park J, Kim M J, Hwang I, Park Y, Kim J Y. CRISPR/Cas9-mediated generation of pathogen- resistant tomato against Tomato yellow leaf curl virus and powdery mildew. Int J Mol Sci, 2021, 22: 1878.
doi: 10.3390/ijms22041878
[13] Qi X, Zhang C, Zhu J, Liu C, Huang C, Li X, Xie C. Genome editing enables next-generation hybrid seed production technology. Mol Plant, 2020, 13: 1262-1269.
doi: 10.1016/j.molp.2020.06.003
[14] Ma X, Zhu Q, Chen Y, Liu Y G. CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant, 2016, 9: 961-974.
doi: 10.1016/j.molp.2016.04.009
[15] Schmutz J, Cannon S B, Schlueter J, Ma J, Mitros T, Nelson W, Hyten D L, Song Q, Thelen J J, Cheng J, Xu D, Hellsten U, May G D, Yu Y, Sakurai T, Umezawa T, Bhattacharyya M K, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang X C, Shinozaki K, Nguyen H T, Wing R A, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker R C, Jackson S A. Genome sequence of the palaeopolyploid soybean. Nature, 2010, 463: 178-183.
doi: 10.1038/nature08670
[16] Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA, 2015, 12: 3570-3575.
[17] Bollier N, Andrade Buono R, Jacobs T B, Nowack M K. Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR. Plant Biotechnol J, 2021, 19: 651-653.
doi: 10.1111/pbi.13525
[18] 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, 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.
doi: 10.1016/j.molp.2015.04.007
[19] Bao A, Chen H, Chen L, Chen S, Hao Q, Guo W, Qiu D, Shan Z, Yang Z, Yuan S, Zhang C, Zhang X, Liu B, Kong F, Li X, Zhou X, Tran L P, Cao D. CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean. BMC Plant Biol, 2019, 19: 131.
doi: 10.1186/s12870-019-1746-6
[20] Cheng Q, Dong L, Su T, Li T, Gan Z, Nan H, Lu S, Fang C, Kong L, Li H, Hou Z, Kou K, Tang Y, Lin X, Zhao X, Chen L, Liu B, Kong F. CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean. BMC Plant Biol, 2019, 19: 562.
doi: 10.1186/s12870-019-2145-8 pmid: 31852439
[21] Di Y H, Sun X J, Hu Z, Jiang Q Y, Song G H, Zhang B, Zhao S S, Zhang H. Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean. Biochem Biophys Res Commun, 2019, 519: 819-823.
doi: 10.1016/j.bbrc.2019.09.074
[22] Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, Xi Y. Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep, 2015, 5: 10342.
doi: 10.1038/srep10342
[23] Liu W, Zhou Y, Li X, Wang X, Dong Y, Wang N, Liu X, Chen H, Yao N, Cui X, Jameel A, Wang F, Li H. Tissue-specific regulation of Gma-miR396 family on coordinating development and low water availability responses. Front Plant Sci, 2017, 8: 1112.
doi: 10.3389/fpls.2017.01112
[24] Niu F N, Jiang Q Y, Sun X J, Hu Z, Wang L X, Zhang H. Large DNA fragment deletion in lncRNA77580 regulates neighboring gene expression in soybean (Glycine max). Funct Plant Biol, 2021, 48: 1139-1147.
doi: 10.1071/FP20400
[25] Zhang X H, Tee L Y, Wang X G, Huang Q S, Yang S H. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol Theor Nucl Acids, 2015, 4: e264.
[26] Cai Y, Chen L, Sun S, Wu C, Yao W, Jiang B, Han T, Hou W. CRISPR/Cas9-mediated deletion of large genomic fragments in soybean. Int J Mol Sci, 2018, 19: 3835.
doi: 10.3390/ijms19123835
[27] Zhang P P, Du H Y, Wang J, Pu Y X, Yang C Y, Yan R J, Yang H, Cheng H, Yu D Y. Mutiplex CRISPR/Cas9-mediated metabolic engineering increases soybean isoflavone content and resistance to soybean mosaic virus. Plant Biotechnol J, 2020, 18: 1384-1395.
doi: 10.1111/pbi.13302
[28] Luo Y, Na R, Nowak J S. Qiu Y, Lu Q S, Yang C, Marsolais F, Tian L. Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing. BMC Plant Biol, 2021, 21: 419.
doi: 10.1186/s12870-021-03138-8
[29] Ye F, Signer E R. RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration. Proc Natl Acad Sci USA, 1996, 93: 10881-10886.
doi: 10.1073/pnas.93.20.10881
[30] Rosser J M, An W. Repeat-induced gene silencing of L1 transgenes is correlated with differential promoter methylation. Gene, 2010, 456: 15-23.
doi: 10.1016/j.gene.2010.02.005 pmid: 20167267
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