CRISPR/Cas9 for Mutagenesis in Rice

  • Si Nian Char
  • Riqing Li
  • Bing YangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1864)


CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) provides a workhorse for genome editing biotechnology. CRISPR/Cas9 tailored for enabling genome editing has been extensively interrogated and widely utilized for precise genomic alterations in eukaryotic organisms including in plant species. The technology holds the great promise to better understand gene functions, elucidate networks, and improve the performance of crop plants such as increasing grain yields, improving nutritional content, and better combating the biotic and abiotic stresses. Various methods or protocols specific for different plant species have been established. Here, we present a CRISPR/Cas9-mediated genome editing protocol in rice, including detailed information about single-guide RNA design, vector construction, plant transformation, and mutant screening processes.

Key words

CRISPR/Cas9 Genome editing Rice Agrobacterium-mediated rice transformation Targeted mutagenesis 



The authors acknowledge the funding support from National Science Foundation (IOS-1238189 to BY), the National Institute of Food and Agriculture of the US Department of Agriculture (2014-67013-21720 to BY).


  1. 1.
    Barrangou R (2013) CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 4:267–278CrossRefGoogle Scholar
  2. 2.
    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
  3. 3.
    Tsai S, Joung J (2016) Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet 17:300–312CrossRefGoogle Scholar
  4. 4.
    Nishimasu H, Ran FA, Hsu PD, Konermann S et al (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156:935–949CrossRefGoogle Scholar
  5. 5.
    Baltes NJ, Voytas DF (2015) Enabling plant synthetic biology through genome engineering. Trends Biotechnol 33:120–131CrossRefGoogle Scholar
  6. 6.
    Hsu P, Lander E, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278CrossRefGoogle Scholar
  7. 7.
    Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42:10903–10914CrossRefGoogle Scholar
  8. 8.
    Brazelton VA, Zarecor S, Wright DA et al (2015) A quick guide to CRISPR sgRNA design tools. GM Crops Food 6:266–276CrossRefGoogle Scholar
  9. 9.
    Hiei Y, Ohta S, Komari T et al (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282CrossRefGoogle Scholar
  10. 10.
    Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Genetics, Development and Cell BiologyIowa State UniversityAmesUSA

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