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Rice Genome Engineering and Gene Editing pp 95–113Cite as

Rapid Vector Construction and Assessment of BE3 and Target-AID C to T Base Editing Systems in Rice Protoplasts

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2238))

Abstract

CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.

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Acknowledgments

This work was supported by grants to Y.Q. from NSF (IOS-1758745), USDA-NIFA (2018-33522-28789), FFAR (593603), and Syngenta Biotechnology and by grants to Y.Z. from the Sichuan Youth Science and Technology Foundation (2017JQ0005), the National Science Foundation of China (31771486), the National Transgenic Major Project (2018ZX08022001-003), and the Fundamental Research Funds for the Central Universities (ZYGX2016J119 and ZYGX2016J122).

Author information

Authors and Affiliations

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

    Simon Sretenovic, Changtian Pan & Yiping Qi

  2. Department of Biotechnology, Center for Informational Biology, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China

    Xu Tang & Yong Zhang

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

    Yiping Qi

Authors
  1. Simon Sretenovic
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  2. Changtian Pan
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  3. Xu Tang
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  4. Yong Zhang
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  5. Yiping Qi
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Corresponding author

Correspondence to Yiping Qi .

Editor information

Editors and Affiliations

  1. Synthetic Biology, Biofuel and Genome Editing R&D, Reliance Industries Ltd, Navi Mumbai, Maharashtra, India

    Anindya Bandyopadhyay

  2. Crop Improvement and Genetics Research Unit, USDA-ARS, Albany, CA, USA

    Roger Thilmony

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Sretenovic, S., Pan, C., Tang, X., Zhang, Y., Qi, Y. (2021). Rapid Vector Construction and Assessment of BE3 and Target-AID C to T Base Editing Systems in Rice Protoplasts. In: Bandyopadhyay, A., Thilmony, R. (eds) Rice Genome Engineering and Gene Editing. Methods in Molecular Biology, vol 2238. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1068-8_7

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  • DOI: https://doi.org/10.1007/978-1-0716-1068-8_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1067-1

  • Online ISBN: 978-1-0716-1068-8

  • eBook Packages: Springer Protocols

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