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Efficient Multiplexed CRISPR-Cas12a Genome Editing in Plants

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CRISPR-Cas Methods

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

Cas12a (formerly Cpf1) is a Class 2 Type V-A clustered regularly interspaced short palindrome repeats (CRISPR ) system that has been widely used in plant genome editing to target AT-rich regions. Cas12a only requires short CRISPR RNAs (crRNAs) for DNA targeting, making it an ideal platform to achieve multiplexed genome engineering. Highly efficient multiplexed genome editing will allow gene family knockout for reverse genetics, manipulation of metabolic pathways, and the simultaneous introduction of multiple agronomically important traits into elite crop cultivars. To apply multiplexed genome editing in plants, here we describe a highly efficient CRISPR-Cas12a expression system and a user-friendly toolbox for vector assembly. In this system, both Cas12a and crRNAs are driven by Pol II promoters, and each crRNA is flanked by hammer head (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise processing. This multiplex system is highly flexible, allowing researchers to make modifications based on plant species and project objectives. The use of this multiplexing toolbox will broaden the application of CRISPR-Cas12a in basic and translational research in plants.

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Acknowledgments

This work was supported by the National Science Foundation Plant Genome Research Program (award no. IOS-1758745 and IOS-2029889), the U.S. Department of Agriculture Biotechnology Risk Assessment Grant Program (award no. 2020-33522-32274), Foundation for Food and Agriculture Research grant (award no. 593603), and Syngenta.

Author information

Authors and Affiliations

  1. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, USA

    Yingxiao Zhang & Yiping Qi

  2. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA

    Yiping Qi

Authors
  1. Yingxiao Zhang
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  2. Yiping Qi
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Corresponding author

Correspondence to Yiping Qi .

Editor information

Editors and Affiliations

  1. Institute of Biotechnology and Genetic Engineering (IBGE), Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

    M. Tofazzal Islam

  2. ICAR-National Rice Research Institute, Cuttack, Odisha, India

    Kutubuddin Ali Molla

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Zhang, Y., Qi, Y. (2021). Efficient Multiplexed CRISPR-Cas12a Genome Editing in Plants. In: Islam, M.T., Molla, K.A. (eds) CRISPR-Cas Methods. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1657-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1657-4_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1656-7

  • Online ISBN: 978-1-0716-1657-4

  • eBook Packages: Springer Protocols

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