A CRISPR/Cas9 Protocol for Target Gene Editing in Barley

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Plant Genome Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2653))

Abstract

Previous studies of gene function rely on the existing natural genetic variation or on induction of mutations by physical or chemical mutagenesis. The availability of alleles in nature, and random mutagenesis induced by physical or chemical means, limits the depth of research. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system provides the means to rapidly modify genomes in a precise and predictable way, making it possible to modulate gene expression and modify the epigenome. Barley is the most appropriate model species for functional genomic analysis of common wheat. Therefore, the genome editing system of barley is very important for the study of wheat gene function. Here we detail a protocol for barley gene editing. The effectiveness of this method has been confirmed in our previous published studies.

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References

  1. Bak RO, Gomez-Ospina N, Porteus MH (2018) Gene editing on center stage. Trends Genet 34(8):600–611

    Article  CAS  PubMed  Google Scholar 

  2. Woolf TM (1998) Therapeutic repair of mutated nucleic acid sequences. Nat Biotechnol 16(4):341–344

    Article  CAS  PubMed  Google Scholar 

  3. 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 

  4. **ng HL, Dong L, Wang ZP et al (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14(1):327

    Article  PubMed  PubMed Central  Google Scholar 

  5. Santamaria ME, Diaz-Mendoza M, Perez-Herguedas D et al (2018) Overexpression of HvIcy6 in barley enhances resistance against Tetranychus urticae and entails partial transcriptomic reprogramming. Int J Mol Sci 19(3):697

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ferdous J, Whitford R, Nguyen M et al (2017) Drought-inducible expression of Hv-miR827 enhances drought tolerance in transgenic barley. Funct Integr Genomics 17(2–3):279–292

    Article  CAS  PubMed  Google Scholar 

  7. Chen J, Liu C, Shi B et al (2017) Overexpression of HvHGGT enhances tocotrienol levels and antioxidant activity in Barley. J Agric Food Chem 65(25):5181–5187

    Article  CAS  PubMed  Google Scholar 

  8. Harwood WA (2014) A protocol for high-throughput Agrobacterium-mediated barley transformation. Methods Mol Biol 1099:251–260

    Article  CAS  PubMed  Google Scholar 

  9. Yang Q, Li S, Li X et al (2019) Expression of the high molecular weight glutenin 1Ay gene from Triticum urartu in barley. Transgenic Res 28(2):225–235

    Article  CAS  PubMed  Google Scholar 

  10. Hinchliffe A, Harwood WA (2019) Agrobacterium-mediated transformation of barley immature embryos. Methods Mol Biol 1900:115–126

    Article  CAS  PubMed  Google Scholar 

  11. Magoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27(21):2957–2963

    Article  PubMed  PubMed Central  Google Scholar 

  12. Jia H, Zhang Y, Orbović V et al (2017) Genome editing of the disease susceptibility gene Cs LOB 1 in citrus confers resistance to citrus canker. Plant Biotechnol J 15(7):817–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Semenova E, Jore MM, Datsenko KA et al (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci 108(25):10098–10103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wiedenheft B, van Duijn E, Bultema JB et al (2011) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci 108(25):10092–10097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25(2–3):179–188

    Article  CAS  PubMed  Google Scholar 

  16. Wang MB, Waterhouse PM (1997) A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes. Plant Mol Biol Rep 15(3):209–215

    Article  CAS  Google Scholar 

  17. Naito Y, Hino K, Bono H et al (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123

    Google Scholar 

  18. Yang Q, Ding JJ, Feng XQ et al (2022) Editing of the starch synthase IIa gene led to transcriptomic andmetabolomic changes and high amylose starch in barley. Carbohydr Polym 285:119238

    Google Scholar 

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Correspondence to Qiantao Jiang .

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Jiang, Q., Yang, Q., Harwood, W., Tang, H., Wei, Y., Zheng, Y. (2023). A CRISPR/Cas9 Protocol for Target Gene Editing in Barley. In: Yang, B., Harwood, W., Que, Q. (eds) Plant Genome Engineering. Methods in Molecular Biology, vol 2653. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3131-7_18

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  • DOI: https://doi.org/10.1007/978-1-0716-3131-7_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3130-0

  • Online ISBN: 978-1-0716-3131-7

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