Advertisement

An Agrobacterium-Mediated CRISPR/Cas9 Platform for Genome Editing in Maize

  • Keunsub Lee
  • Huilan Zhu
  • Bing Yang
  • Kan Wang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

Precise genome engineering can be efficiently made using the revolutionary tool named CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) systems. Adapted from the bacterial immune system, CRISPR/Cas systems can generate highly specific double-strand breaks (DSBs) at the target site, and desired sequence modifications can be introduced during the DSB repair process, such as nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. CRISPR/Cas9 is the most widely used genome editing tool for targeted mutagenesis, precise sequence modification, transcriptional reprogramming, epigenome editing, disease treatment, and many more. The ease of use and high specificity make CRISPR/Cas9 a great tool not only for basic researches but also for crop trait improvements, such as higher grain yield, better tolerance to abiotic stresses, enhanced disease resistance, and better nutritional contents. In this protocol, we present a step-by-step guide to the CRISPR/Cas9-mediated targeted mutagenesis in maize Hi II genotype. Detailed procedures will guide through the essential steps including gRNA design, CRISPR/Cas9 vector construction, Agrobacterium-mediated maize immature embryo transformation, and molecular analysis of the transgenic plants to identify desired mutant lines.

Key words

Agrobacterium-mediated transformation CRISPR/Cas9 Genome editing Maize Targeted mutagenesis 

Notes

Acknowledgments

The authors wish to thank Marcy Main, Daniel Little, and Minjeong Kang for their contributions to this work. This project was partially supported by the US National Science Foundation (BREAD #1543888 to K.W. and B.Y.); by the USDA National Institute of Food and Agriculture, Hatch project number # IOW05162; by the State of Iowa funds; and by the Crop Bioengineering Center of Iowa State University.

References

  1. 1.
    Kim H, Kim ST, Kim SG, Kim JS (2015) Targeted genome editing for crop improvement. Plant Breed Biotechnol 3:283–290CrossRefGoogle Scholar
  2. 2.
    Yang N, Wang R, Zhao Y (2017) Revolutionize genetic studies and crop improvement with high-through and genome-scale CRISPR/Cas9 gene editing technology. Mol Plant 10:1141–1143CrossRefGoogle Scholar
  3. 3.
    Gao C (2018) The future of CRISPR technologies in agriculture. Nat Rev 19:275–276CrossRefGoogle Scholar
  4. 4.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefGoogle Scholar
  5. 5.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefGoogle Scholar
  6. 6.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefGoogle Scholar
  7. 7.
    Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK (2016) High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495CrossRefGoogle Scholar
  8. 8.
    Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F (2016) Rationally engineered Cas9 nucleases with improved specificity. Science 351:84–88CrossRefGoogle Scholar
  9. 9.
    Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–451CrossRefGoogle Scholar
  10. 10.
    Mali P, Esvelt KM, Church GM (2013b) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963CrossRefGoogle Scholar
  11. 11.
    Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD (2014) A protein tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159:635–646CrossRefGoogle Scholar
  12. 12.
    Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H et al (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588CrossRefGoogle Scholar
  13. 13.
    Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ et al (2015) Highly-efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328CrossRefGoogle Scholar
  14. 14.
    Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPRCas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344CrossRefGoogle Scholar
  15. 15.
    Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L (2016) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol Plant 9:628–631CrossRefGoogle Scholar
  16. 16.
    Zhang JP, Li XL, Li GH, Chen W, Arakaki C, Botimer GD, Baylink D, Zhang L, Wen W, Fu YW et al (2017) Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol 18:35CrossRefGoogle Scholar
  17. 17.
    Hilton IB, D’Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA (2015) Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33:510–517CrossRefGoogle Scholar
  18. 18.
    Thakore PI, D’Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, Reddy TE, Crawford GE, Gersbach CA (2015) Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12:1143–1149CrossRefGoogle Scholar
  19. 19.
    Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424CrossRefGoogle Scholar
  20. 20.
    Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY et al (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353:aaf8729CrossRefGoogle Scholar
  21. 21.
    Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR (2017) Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol 35:371–376CrossRefGoogle Scholar
  22. 22.
    Kuscu C, Parlak M, Tufan T, Yang J, Szlachta K, Wei X, Mammadov R, Adli M (2017) CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat Methods 14:710–712CrossRefGoogle Scholar
  23. 23.
    Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491CrossRefGoogle Scholar
  24. 24.
    Deng W, Shi X, Tjian R, Lionnet T, Singer RH (2015) CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc Natl Acad Sci U S A 112:11870–11875CrossRefGoogle Scholar
  25. 25.
    Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T (2016) Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat Biotechnol 34:528–530CrossRefGoogle Scholar
  26. 26.
    Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13:659–662CrossRefGoogle Scholar
  27. 27.
    Yin H, Wen X, Chen S, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 32:551–553CrossRefGoogle Scholar
  28. 28.
    Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, Xie Z, Bai M, Yin Q, Liang D (2015) Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res 25:67–79CrossRefGoogle Scholar
  29. 29.
    Morgens DW, Wainberg M, Boyle EA, Orsu O, Araya CL, Tsui CK, Haney MS, Hess GT, Han K, Jeng EE et al (2017) Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat Commun 8:15178CrossRefGoogle Scholar
  30. 30.
    Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161:1169–1175PubMedPubMedCentralGoogle Scholar
  31. 31.
    Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169:931–945CrossRefGoogle Scholar
  32. 32.
    Feng C, Yuan J, Wang R, Liu Y, Birchler JA, Han F (2016) Efficient targeted genome modification in maize using CRISPR/Cas9 system. J Genet Genomics 43:37–43CrossRefGoogle Scholar
  33. 33.
    Zhu J, Song N, Sun S, Yang W, Zhao H, Song W, Lai J (2016) Efficiency and inheritance of targeted mutagenesis in maize using CRISPR-Cas9. J Genet Genomics 43:25–36CrossRefGoogle Scholar
  34. 34.
    Char SN, Neelakandan AK, Nahanpun H, Frame B, Main M, Spalding MH, Becraft PW, Meyers BC, Walbot V, Wang K, Yang B (2017) An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnol J 15:257–268CrossRefGoogle Scholar
  35. 35.
    Li C, Liu C, Qi X, Wu Y, Fei X, Mao L, Cheng B, Li X, Xie C (2017) RNA-guided Cas9 as an in vivo desired-target mutator in maize. Plant Biotechnol J 15:1566–1576CrossRefGoogle Scholar
  36. 36.
    Feng C, Su H, Han B, Wang R, Liu Y, Guo X, Liu C, Zhang J, Yuan J, Birchler JA, Han F (2018) High efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize. Plant Biotechnol J. https://doi.org/10.1111/pbi.12920CrossRefGoogle Scholar
  37. 37.
    Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301CrossRefGoogle Scholar
  38. 38.
    Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  39. 39.
    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
  40. 40.
    White J, Chang SY, Bibb MJ, Bibb MJ (1990) A cassette containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucleic Acids Res 18:1062CrossRefGoogle Scholar
  41. 41.
    Bertani G (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186:595–600CrossRefGoogle Scholar
  42. 42.
    Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefGoogle Scholar
  43. 43.
    Song T, Toma C, Nakasone N, Iwanaga M (2004) Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria. J Med Microbiol 53:477–482CrossRefGoogle Scholar
  44. 44.
    Brazelton VA Jr, Zarecor S, Wright DA, Wang Y, Liu J, Chen K, Yang B, Lawrence-Dill CJ (2015) A quick guide to CRISPR sgRNA design tools. GM Crops Food 6:266–276CrossRefGoogle Scholar
  45. 45.
    Ding Y, Hong L, Chen LL, Xie K (2016) Recent advances in genome editing using CRISPR/Cas9. Front Plant Sci 7:703. https://doi.org/10.3389/fpls.2016.00703CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Park JP, Bae S, Kim JS (2015) Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31:4014–4016CrossRefGoogle Scholar
  47. 47.
    Andorf CM, Cannon EK, Portwood JL 2nd, Gardiner JM, Harper LC, Schaeffer ML, Braun BL, Campbell DA, Vinnakota AG, Sribalusu VV et al (2016) MaizeGDB update: new tools, data and interface for the maize model organism database. Nucleic Acids Res 44:D1195–D1201CrossRefGoogle Scholar
  48. 48.
    Li JF, Aach J, Norville JE, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated plant genome editing via guide RNA/Cas9. Nat Biotechnol 31:688–691CrossRefGoogle Scholar
  49. 49.
    Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877CrossRefGoogle Scholar
  50. 50.
    Frame B, Warnberg K, Main M, Wang K (2015) Maize (Zea mays L.). In: Wang K (ed) Agrobacterium protocols. Springer, New York, pp 101–117Google Scholar
  51. 51.
    Allen GC, Flores-Vergara MA, Krasynanski S, Kumar S, Thompson WF (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 1:2320–2325CrossRefGoogle Scholar
  52. 52.
    Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42:e168CrossRefGoogle Scholar
  53. 53.
    Liu W, Xie X, Ma X, Li J, Chen J, Liu YG (2015) DSDecode: A web-based tool for decoding sequencing chromatograms for genotyping of targeted mutations. Mol Plant 8:1431–1433CrossRefGoogle Scholar
  54. 54.
    Oltmanns H, Frame B, Lee LY, Johnson S, Li B, Wang K, Gelvin SB (2010) Generation of “backbone” free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. Plant Physiol 152:1158–1166CrossRefGoogle Scholar
  55. 55.
    Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J 3:2723–2730CrossRefGoogle Scholar
  56. 56.
    Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Coop Newsl 65:92–93Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Crop Bioengineering CenterIowa State UniversityAmesUSA

Personalised recommendations