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A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing

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Plant Genome Editing with CRISPR Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1917))

Abstract

The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.

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Acknowledgments

This study was supported by the National Natural Science Foundation of China to KX (31571374 and 31622047) and National Science Foundation Plant Genome Research Project Grant (1740874) to YY. This work was also supported by the USDA National Institute of Food and Agriculture Hatch project PEN04659.

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Authors and Affiliations

  1. National Key Laboratory for Crop Genetic Improvement and Plant Gene Research Center (Wuhan), Huazhong Agricultural University, Wuhan, China

    Kabin Xie

  2. Department of Plant Pathology and Environmental Microbiology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, Pennsylvania, PA, USA

    Yinong Yang

Authors
  1. Kabin Xie
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  2. Yinong Yang
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Corresponding author

Correspondence to Yinong Yang .

Editor information

Editors and Affiliations

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

    Yiping Qi

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Xie, K., Yang, Y. (2019). A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_5

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  • DOI: https://doi.org/10.1007/978-1-4939-8991-1_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8990-4

  • Online ISBN: 978-1-4939-8991-1

  • eBook Packages: Springer Protocols

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