GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana

  • Swapnil S. Thakare
  • Navita Bansal
  • S. Vanchinathan
  • G. Rama Prashat
  • Veda Krishnan
  • Archana Sachdev
  • Shelly PraveenEmail author
  • T. VinuthaEmail author
Original Article


The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9) is simple and highly efficient technology applied to functional studies of genes and genetic crop improvement. In this study, we have demonstrated the utility of green fluorescent protein (GFP) marker to detect the targeting efficiency of gRNAs. As a proof of concept, Glycine max De-Etiolated 1 (GmDET1) gene was chosen and tagged with GFP to rapidly analyze genome editing efficiency of gRNAs. Results showed weaker GFP fluorescence signal in the N. benthamiana leaves co-infiltrated with GmDET1-GFP overexpression (OE) + DET1 gRNA1 constructs as compared to the stronger GFP florescence signal in the leaves co-infiltrated with DET1 gRNA2 and gRNA3 constructs, thus indicating the highest of DET1 gRNA1. These results were further confirmed by the detection of the mutation frequencies through T7 endonuclease (T7E1) assay and sequencing; the highest mutation rate of 38.46% in GmDET1 targeted by DET1 gRNA1 to that of DET1 gRNA2 (7.69%) and gRNA3 (15.38%) was observed. Thus our studies showed “GFP tagging” as the most reliable and rapid method-one can apply to minimize the generation of non-edited transgenic plants resulting from inefficient gRNAs.


CRISPR/Cas9 gRNA GFP fluorescent marker N. benthamiana Glycine max DET1 



De-Etiolated 1


Clustered regularly interspaced short palindromic repeats/CRISPR—associated proteins 9


Green fluorescent protein


T7 Endonuclease I


Guide RNA



We thank the laboratory of Robert Stupar for Cas9 (MDC123) and gRNA shuttle vectors (pBlu/gRNA) (Addgene plasmid # 59184 and 59188 respectively). Permission was taken to conduct this study from Institute Biosafety Committee. The presented research work was carried out with the financial support received from ICAR-IARI, Division of Biochemistry, New Delhi-110012.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

13562_2019_540_MOESM1_ESM.docx (417 kb)
Supplementary material 1 (DOCX 417 kb)


  1. Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475PubMedPubMedCentralCrossRefGoogle Scholar
  2. Belhaj K, Garcia AC, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39PubMedPubMedCentralCrossRefGoogle Scholar
  3. Bomgardner M (2017) CRISPR: a new toolbox for better crops. Chem Eng News 95(24):33–34Google Scholar
  4. Chari R, Mali P, Moosburner M, Church GM (2015) Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods 12(9):823–826PubMedPubMedCentralCrossRefGoogle Scholar
  5. Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6(3):325–330PubMedCrossRefPubMedCentralGoogle Scholar
  6. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–141PubMedPubMedCentralCrossRefGoogle Scholar
  7. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  8. Davuluri GR, Tuinen VA, Fraser PD, Manfredonia A, Newman R, Burgess D, Brummell DA, King SR, Palys J, Uhlig J, Bramley PM, Pennings HM, Bowler C (2005) Fruit-specific RNAi-mediated suppression of DET1 enhances tomato nutritional quality. Nat Biotechnol 23:890–895PubMedCrossRefPubMedCentralGoogle Scholar
  9. Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12):1262–1267PubMedPubMedCentralCrossRefGoogle Scholar
  10. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR Cas9. Science 346(6213):1258096PubMedCrossRefPubMedCentralGoogle Scholar
  11. Dwiyanti MS, Ujiie A, Le TBT, Yamada T, Kitamura K (2007) Genetic analysis of high α-tocopherol content in soybean seeds. Breed Sci 57(1):23–28CrossRefGoogle Scholar
  12. Enfissi EM, Barneche F, Ahmed I, Lichtle C, Gerrish C, McQuinn RP, Giovannoni JJ, Lopez-Juez E, Bowler C, Bramley PM, Fraserm PD (2010) Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 downregulated tomato fruit. Plant Cell 22(4):1190–1215PubMedPubMedCentralCrossRefGoogle Scholar
  13. Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637PubMedCrossRefPubMedCentralGoogle Scholar
  14. Fister AS, Landherr L, Maximova SN, Guiltinan MJ (2018) Transient expression of CRISPR/Cas9 machinery targeting tcnpr3 enhances defense response in Theobroma cacao. Front Plant Sci 9:268PubMedPubMedCentralCrossRefGoogle Scholar
  15. Gagnon JA, Valen E, Thyme SB, Huang P, Akhmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, Schier AF (2014) Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS ONE 9(5):e98186PubMedPubMedCentralCrossRefGoogle Scholar
  16. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109(39):15539–15540CrossRefGoogle Scholar
  17. Hanson MR, Köhler RH (2001) GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 52(356):529–539PubMedCrossRefPubMedCentralGoogle Scholar
  18. Ishii T, Araki M (2016) Consumer acceptance of food crops developed by genome editing. Plant Cell Rep 35(7):1507–1518PubMedCrossRefPubMedCentralGoogle Scholar
  19. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2014) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41(20):e188CrossRefGoogle Scholar
  20. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S (2014) Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343(6176):1247997PubMedPubMedCentralCrossRefGoogle Scholar
  21. Li J, Shou J, Guo Y, Tang Y, Wu Y, Jia Z (2015) Eefficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9. J Mol Cell Biol 7(4):284–298PubMedPubMedCentralCrossRefGoogle Scholar
  22. Lichtenthaler HK (2007) Biosynthesis, accumulation and emission of carotenoids, α-tocopherol, plastoquinone and isoprene in leaves under high photosynthetic irradiance. Photosynth Res 92:163–179PubMedCrossRefPubMedCentralGoogle Scholar
  23. Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31(6):777–786PubMedCrossRefPubMedCentralGoogle Scholar
  24. Liu Y, Roof S, Ye Z, Barry C, Tuinen VA, Verbalov J, Bowler C, Giovannoni J (2004) Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proc Natl Acad Sci USA 101:9897–9902PubMedCrossRefPubMedCentralGoogle Scholar
  25. Liu D, Hu R, Palla KJ, Tuskan GA, Yang X (2016) Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research. Curr Opin Plant Biol 30:70–77PubMedCrossRefPubMedCentralGoogle Scholar
  26. Mao Y, Zhang H, Xu N, Zhang B, Gao F, Zhu JK (2013) Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6(6):2008–2011PubMedPubMedCentralCrossRefGoogle Scholar
  27. Monica F, Sentmanat ST, Florian CP, Jon P, Pruett-Mille SM (2018) A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 8:888CrossRefGoogle Scholar
  28. Nishimasu H, Ran F, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–949PubMedPubMedCentralCrossRefGoogle Scholar
  29. Pan C, Ye L, Qin L, Liu X, He Y, Wang J, Chen L, Lu G (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6:24765PubMedPubMedCentralCrossRefGoogle Scholar
  30. Pepper A, Delaney T, Washburn T, Poole D, Chory J (1994) DET1, a negative regulator of light-mediated development and gene expression in Arabidopsis, encodes a novel nuclear localized protein. Cell 78:109–116PubMedCrossRefPubMedCentralGoogle Scholar
  31. Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, Liu LP, Yang Z, Mao D, Sun L (2013) Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci USA 110:19012–19017PubMedCrossRefPubMedCentralGoogle Scholar
  32. Ren X, Yang Z, Xu J, Sun J, Mao D, Hu Y, Yang SJ, Qiao HH, Wang X, Hu Q, Deng P, Liu LP, Ji JY, Li JB, Ni JQ (2014) Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila. Cell Rep 9(3):1151–1162PubMedPubMedCentralCrossRefGoogle Scholar
  33. Sahana N, Kaur H, Basavaraj Tena F, Jain RK, Palukaitis P, Canto T, Praveen S (2012) Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro. PLoS ONE 7:e52546PubMedPubMedCentralCrossRefGoogle Scholar
  34. Schafer E, Bowle C (2002) Phytochrome-mediated photoperception and signal transduction in higher plants. EMBO Rep 3:1042–1048PubMedPubMedCentralCrossRefGoogle Scholar
  35. Schiml S, Fauser F, Puchta H (2014) The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J 80(6):1139–1150CrossRefGoogle Scholar
  36. Shukla S, Saini P, Smriti Jha S, Ambudkar SV, Prasad R (2003) Functional characterization of Candida albicans ABC transporter Cdr1p. Eukaryot Cell 2:1361–1375PubMedPubMedCentralCrossRefGoogle Scholar
  37. Singh R, Kuscu C, Quinlan A, Qi Y, Adli M (2015) Cas9-chromatin binding information enables more accurate CRISPR off-target prediction. Nucleic Acids Res 43:e118PubMedPubMedCentralCrossRefGoogle Scholar
  38. Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, Sheffield NC, Stergachis AB, Wang H, Vernot B (2012) The accessible chromatin landscape of the human genome. Nature 489:75–82PubMedPubMedCentralCrossRefGoogle Scholar
  39. Tian J, Pei H, Zhang S, Chen J, Chen W, Yang Y, Meng Y, You J, Gao J, Ma N (2014) TRV–GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function. J Exp Bot 65(1):311–322PubMedCrossRefPubMedCentralGoogle Scholar
  40. Tycko J, Myer VE, Hsu PD (2016) Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell 63(3):355–370PubMedPubMedCentralCrossRefGoogle Scholar
  41. Voytas DF, Gao C (2014) Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol 12(6):e1001877PubMedPubMedCentralCrossRefGoogle Scholar
  42. Wang G, Xu Y (2008) Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference. Plant Cell Rep 27(7):1177–1184PubMedCrossRefPubMedCentralGoogle Scholar
  43. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84CrossRefGoogle Scholar
  44. Wei S, Li X, Gruber MY, Li R, Zhou R, Zebarjadi A, Hannoufa A (2009) RNAi-mediated suppression of DET1 alters the levels of carotenoids and sinapate esters in seeds of Brassica napus. J Agric Food Chem 57:5326–5333PubMedCrossRefPubMedCentralGoogle Scholar
  45. Weigel D, Glazebrook J (2006) Transformation of agrobacterium using the freeze-thaw method. CSH Protocols 2006(7):1031–1036Google Scholar
  46. Wong N, Liu W, Wang X (2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16:218PubMedPubMedCentralCrossRefGoogle Scholar
  47. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, Cheng AW, Trevino AE, Konermann S, Chen S (2014) Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32:670–676PubMedPubMedCentralCrossRefGoogle Scholar
  48. Xie S, Shen B, Zhang C, Huang X, Zhang Y (2014) sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS ONE 9:e100448PubMedPubMedCentralCrossRefGoogle Scholar
  49. Xu H, Xiao T, Chen CH, Li W, Meyer C, Wu Q (2015) Sequence determinants of improved CRISPR sgRNA design. Genome Res 25(8):1147–1157PubMedPubMedCentralCrossRefGoogle Scholar
  50. Zhang H, Zhang J, Wei P, Zhang B, Gou F, Feng Z (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807PubMedCrossRefPubMedCentralGoogle Scholar
  51. Zheng X, Yang S, Zhang D, Zhong Z, Tang X, Deng K (2016) Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. Plant Cell Rep 35(7):1545–1554PubMedCrossRefPubMedCentralGoogle Scholar
  52. Zhu H, Liang C (2018) CRISPR-DT: designing gRNAs for the CRISPR-Cpf1system with improved target efficiency and specificity. bioRxiv: 269910Google Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2019

Authors and Affiliations

  • Swapnil S. Thakare
    • 1
  • Navita Bansal
    • 1
  • S. Vanchinathan
    • 1
  • G. Rama Prashat
    • 2
  • Veda Krishnan
    • 1
  • Archana Sachdev
    • 1
  • Shelly Praveen
    • 1
    Email author
  • T. Vinutha
    • 1
    Email author
  1. 1.Division of BiochemistryIARINew DelhiIndia
  2. 2.Division of GeneticsIARINew DelhiIndia

Personalised recommendations