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

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Abstract

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.

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Abbreviations

DET1:

De-Etiolated 1

CRISPR/Cas9:

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

GFP:

Green fluorescent protein

T7E1:

T7 Endonuclease I

gRNA:

Guide RNA

References

  • 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 30:1473–1475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 9:39

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bomgardner M (2017) CRISPR: a new toolbox for better crops. Chem Eng News 95(24):33–34

    Google Scholar 

  • 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):823–826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):325–330

    Article  CAS  PubMed  Google Scholar 

  • 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 24:132–141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 23:890–895

    Article  CAS  PubMed  Google Scholar 

  • 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):1262–1267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR Cas9. Science 346(6213):1258096

    Article  PubMed  CAS  Google Scholar 

  • 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):23–28

    Article  CAS  Google Scholar 

  • 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):1190–1215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 111:4632–4637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 9:268

    Article  PubMed  PubMed Central  Google Scholar 

  • 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):e98186

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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):15539–15540

    Article  CAS  Google Scholar 

  • Hanson MR, Köhler RH (2001) GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 52(356):529–539

    Article  CAS  PubMed  Google Scholar 

  • Ishii T, Araki M (2016) Consumer acceptance of food crops developed by genome editing. Plant Cell Rep 35(7):1507–1518

    Article  CAS  PubMed  Google Scholar 

  • 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):e188

    Article  CAS  Google Scholar 

  • 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):1247997

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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):284–298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lichtenthaler HK (2007) Biosynthesis, accumulation and emission of carotenoids, α-tocopherol, plastoquinone and isoprene in leaves under high photosynthetic irradiance. Photosynth Res 92:163–179

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31(6):777–786

    Article  CAS  PubMed  Google Scholar 

  • 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 101:9897–9902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 30:70–77

    Article  CAS  PubMed  Google Scholar 

  • 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):2008–2011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Monica F, Sentmanat ST, Florian CP, Jon P, Pruett-Mille SM (2018) A survey of validation strategies for CRISPR-Cas9 editing. Sci Rep 8:888

    Article  CAS  Google Scholar 

  • 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 156:935–949

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 6:24765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 78:109–116

    Article  CAS  PubMed  Google Scholar 

  • 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 110:19012–19017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):1151–1162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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:e52546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schafer E, Bowle C (2002) Phytochrome-mediated photoperception and signal transduction in higher plants. EMBO Rep 3:1042–1048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):1139–1150

    Article  CAS  PubMed  Google Scholar 

  • Shukla S, Saini P, Smriti Jha S, Ambudkar SV, Prasad R (2003) Functional characterization of Candida albicans ABC transporter Cdr1p. Eukaryot Cell 2:1361–1375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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:e118

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 489:75–82

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):311–322

    Article  CAS  PubMed  Google Scholar 

  • Tycko J, Myer VE, Hsu PD (2016) Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell 63(3):355–370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Voytas DF, Gao C (2014) Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol 12(6):e1001877

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang G, Xu Y (2008) Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference. Plant Cell Rep 27(7):1177–1184

    Article  PubMed  CAS  Google Scholar 

  • Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84

    Article  CAS  PubMed  Google Scholar 

  • 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 57:5326–5333

    Article  CAS  PubMed  Google Scholar 

  • Weigel D, Glazebrook J (2006) Transformation of agrobacterium using the freeze-thaw method. CSH Protocols 2006(7):1031–1036

    Google Scholar 

  • Wong N, Liu W, Wang X (2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16:218

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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 32:670–676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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:e100448

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu H, Xiao T, Chen CH, Li W, Meyer C, Wu Q (2015) Sequence determinants of improved CRISPR sgRNA design. Genome Res 25(8):1147–1157

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 12:797–807

    Article  CAS  PubMed  Google Scholar 

  • 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):1545–1554

    Article  CAS  PubMed  Google Scholar 

  • Zhu H, Liang C (2018) CRISPR-DT: designing gRNAs for the CRISPR-Cpf1system with improved target efficiency and specificity. bioRxiv: 269910

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Acknowledgements

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.

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Authors and Affiliations

  1. Division of Biochemistry, IARI, New Delhi, 110012, India

    Swapnil S. Thakare, Navita Bansal, S. Vanchinathan, Veda Krishnan, Archana Sachdev, Shelly Praveen & T. Vinutha

  2. Division of Genetics, IARI, New Delhi, 110012, India

    G. Rama Prashat

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  1. Swapnil S. Thakare
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  2. Navita Bansal
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Correspondence to Shelly Praveen or T. Vinutha.

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Thakare, S.S., Bansal, N., Vanchinathan, S. et al. GFP tagging based method to analyze the genome editing efficiency of CRISPR/Cas9-gRNAs through transient expression in N. benthamiana. J. Plant Biochem. Biotechnol. 29, 183–192 (2020). https://doi.org/10.1007/s13562-019-00540-0

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