Science Bulletin

, Volume 60, Issue 15, pp 1332–1347 | Cite as

A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana

  • Wenshan Liu
  • Xiaohong Zhu
  • Mingguang Lei
  • Qingyou XiaEmail author
  • Jose Ramon Botella
  • Jian-Kang Zhu
  • Yanfei MaoEmail author
Article Life & Medical Sciences


The newly developed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations and corrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA (sgRNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sgRNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.


CRISPR/Cas9 Targeted gene editing Genome engineering Arabidopsis thaliana 


CRISPR是一类来源于细菌的“规律间隔成簇短回文重复序列”,通过与Cas9蛋白形成二元复合体来识别特定的DNA序列。近年来的研究表明,经过改造的CRISPR/Cas9系统可以在植物体内实现对目标基因的高效编辑,从而有望取代经典的基因突变技术和转基因技术来满足基因功能研究和作物品种研发的需要。定制后的CRISPR/Cas9复合体可以根据给定的序列,以碱基配对的方式结合到目标位点上,并造成双链DNA断裂。这种严重的DNA损伤,会激活细胞内源的DNA损伤修复通路,在缺乏模板的情况下,这类修复往往是不正确的,极易导致断裂位置的碱基改变。本文详细描述了如何利用已有的CRISPR/Cas9系统,在拟南芥中进行可遗传的基因定点修饰的策略和具体步骤, 包括靶位点的选择、引导RNA的设计、载体构建和转基因植物的检测和分析。另外,我们也提供了在原有质粒的基础上,构建双敲载体的方法。本实验方案所涉及的原理和策略也同样可以移植到其他植物品种上,来实现可遗传的基因修饰。



The work was supported by the Chinese Academy of Sciences and China Scholarship Council (201206050103).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11434_2015_848_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 16 kb)


  1. 1.
    Redondo P, Prieto J, Munoz IG et al (2008) Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456:107–111CrossRefGoogle Scholar
  2. 2.
    Lloyd A (2005) Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci USA 102:2232–2237CrossRefGoogle Scholar
  3. 3.
    Wright DA, Townsend JA, Winfrey RJ Jr et al (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705CrossRefGoogle Scholar
  4. 4.
    Shukla VK, Doyon Y, Miller JC et al (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437–441CrossRefGoogle Scholar
  5. 5.
    Zhang F, Voytas DF (2010) Targeted mutagenesis in Arabidopsis using zinc-finger nucleases. Methods Mol Biol 701:167–177CrossRefGoogle Scholar
  6. 6.
    Miller JC, Holmes MC, Wang J et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785CrossRefGoogle Scholar
  7. 7.
    Miller JC, Tan S, Qiao G et al (2010) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148CrossRefGoogle Scholar
  8. 8.
    Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846CrossRefGoogle Scholar
  9. 9.
    Mahfouz MM, Li L, Shamimuzzaman M et al (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108:2623–2628CrossRefGoogle Scholar
  10. 10.
    Li T, Liu B, Spalding MH et al (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotechnol 30:390–392CrossRefGoogle Scholar
  11. 11.
    Christian M, Qi Y, Zhang Y et al (2013) Targeted mutagenesis of Arabidopsis thaliana using engineered TAL effector nucleases. G3 (Bethesda) 3:1697–1705CrossRefGoogle Scholar
  12. 12.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefGoogle Scholar
  13. 13.
    Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefGoogle Scholar
  14. 14.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefGoogle Scholar
  15. 15.
    Sung P, Klein H (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7:739–750CrossRefGoogle Scholar
  16. 16.
    Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211CrossRefGoogle Scholar
  17. 17.
    Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271CrossRefGoogle Scholar
  18. 18.
    Garneau JE, Dupuis ME, Villion M et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71CrossRefGoogle Scholar
  19. 19.
    Marraffini LA, Sontheimer EJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463:568–571CrossRefGoogle Scholar
  20. 20.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096CrossRefGoogle Scholar
  21. 21.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278CrossRefGoogle Scholar
  22. 22.
    Feng Z, Zhang B, Ding W et al (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23:1229–1232CrossRefGoogle Scholar
  23. 23.
    Jiang W, Zhou H, Bi H et al (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:e188CrossRefGoogle Scholar
  24. 24.
    Li JF, Norville JE, Aach J et al (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691CrossRefGoogle Scholar
  25. 25.
    Mao Y, Zhang H, Xu N et al (2013) Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6:2008–2011CrossRefGoogle Scholar
  26. 26.
    Miao J, Guo D, Zhang J et al (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23:1233–1236CrossRefGoogle Scholar
  27. 27.
    Nekrasov V, Staskawicz B, Weigel D et al (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693CrossRefGoogle Scholar
  28. 28.
    Shan Q, Wang Y, Li J et al (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688CrossRefGoogle Scholar
  29. 29.
    Upadhyay SK, Kumar J, Alok A et al (2013) RNA-guided genome editing for target gene mutations in wheat. G3 (Bethesda) 3:2233–2238CrossRefGoogle Scholar
  30. 30.
    Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6:1975–1983CrossRefGoogle Scholar
  31. 31.
    Brooks C, Nekrasov V, Lippman ZB et al (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated system. Plant Physiol 166:1292–1297CrossRefGoogle Scholar
  32. 32.
    Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359CrossRefGoogle Scholar
  33. 33.
    Jia H, Wang N (2014) Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One 9:e93806CrossRefGoogle Scholar
  34. 34.
    Xing HL, Dong L, Wang ZP et al (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327CrossRefGoogle Scholar
  35. 35.
    Zhang H, Zhang J, Wei P et al (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807CrossRefGoogle Scholar
  36. 36.
    Feng Z, Mao Y, Xu N et al (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637CrossRefGoogle Scholar
  37. 37.
    Yan M, Zhou SR, Xue HW (2014) CRISPR primer designer: design primers for knockout and chromosome imaging CRISPR-Cas system. J Integr Plant Biol. doi: 10.1111/jipb.12295 Google Scholar
  38. 38.
    Weigel D, Glazebrook J (2006) Transformation of Agrobacterium using the freeze-thaw method. CSH Protoc. doi: 10.1101/pdb.prot4666 Google Scholar
  39. 39.
    Weigel D, Glazebrook J (2006) In planta transformation of Arabidopsis. CSH Protoc. doi: 10.1101/pdb.prot4668 Google Scholar
  40. 40.
    Wu FH, Shen SC, Lee LY et al (2009) Tape-Arabidopsis Sandwich: a simpler Arabidopsis protoplast isolation method. Plant Methods 5:16CrossRefGoogle Scholar
  41. 41.
    Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefGoogle Scholar
  42. 42.
    Springer NM (2010) Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB. CSH Protoc. doi: 10.1101/pdb.prot5515 Google Scholar
  43. 43.
    Neff MM, Neff JD, Chory J et al (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J 14:387–392CrossRefGoogle Scholar
  44. 44.
    Qiu P, Shandilya H, D’alessio JM et al (2004) Mutation detection using Surveyor nuclease. Biotechniques 36:702–707Google Scholar
  45. 45.
    Xiao A, Wang Z, Hu Y et al (2013) Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 41:e141CrossRefGoogle Scholar
  46. 46.
    Canver MC, Bauer DE, Dass A et al (2014) Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J Biol Chem 289:21312–21324CrossRefGoogle Scholar
  47. 47.
    Jiang W, Bikard D, Cox D et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239CrossRefGoogle Scholar
  48. 48.
    Wang T, Wei JJ, Sabatini DM et al (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84CrossRefGoogle Scholar
  49. 49.
    O’malley RC, Ecker JR (2010) Linking genotype to phenotype using the Arabidopsis unimutant collection. Plant J 61:928–940CrossRefGoogle Scholar
  50. 50.
    Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964CrossRefGoogle Scholar
  51. 51.
    Wu X, Kriz AJ, Sharp PA (2015) Target specificity of the CRISPR-Cas9 system. Quant Biol 2:59–70CrossRefGoogle Scholar
  52. 52.
    Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–3575CrossRefGoogle Scholar
  53. 53.
    Baltes NJ, Gil-Humanes J, Cermak T et al (2014) DNA replicons for plant genome engineering. Plant Cell 26:151–163CrossRefGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Wenshan Liu
    • 1
    • 2
    • 4
  • Xiaohong Zhu
    • 2
  • Mingguang Lei
    • 3
  • Qingyou Xia
    • 4
    Email author
  • Jose Ramon Botella
    • 5
  • Jian-Kang Zhu
    • 2
    • 3
  • Yanfei Mao
    • 2
    Email author
  1. 1.School of Life SciencesChongqing UniversityChongqingChina
  2. 2.Shanghai Center for Plant Stress Biology, Shanghai Institutes of Biological SciencesChinese Academy of SciencesShanghaiChina
  3. 3.Department of Horticulture and Landscape ArchitecturePurdue UniversityWest LafayetteUSA
  4. 4.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina
  5. 5.School of Agriculture and Food SciencesUniversity of QueenslandBrisbaneAustralia

Personalised recommendations