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Plant Gene Regulation Using Multiplex CRISPR-dCas9 Artificial Transcription Factors

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Maize

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1676))

Abstract

Besides genome editing, the CRISPR-Cas9-based platform provides a new way of engineering artificial transcription factors (ATFs). Multiplex of guide RNA (gRNA) expression cassettes holds a great promise for many useful applications of CRISPR-Cas9. In this chapter, we provide a detailed protocol for building advanced multiplexed CRISPR-dCas9-Activator/repressor T-DNA vectors for carrying out transcriptional activation or repression experiments in plants. We specifically describe the assembly of multiplex T-DNA vectors that can express multiple gRNAs to activate a silenced gene, or to repress two independent miRNA genes simultaneously in Arabidopsis. We then describe a “higher-order” vector assembly method for increased multiplexing capacity. This higher-order assembly method in principle allows swift stacking of gRNAs cassettes that are only limited by the loading capacity of a cloning or expression vector.

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Acknowledgments

This work is supported by a Collaborative Funding Grant from North Carolina Biotechnology Center and Syngenta (2016-CFG-8003) to Y.Q.

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Authors and Affiliations

  1. Department of Biology, East Carolina University, Howell Science Complex, Greenville, NC, 27858, USA

    Levi G. Lowder

  2. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, 20742, USA

    Aimee Malzahn & Yiping Qi

Authors
  1. Levi G. Lowder
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  2. Aimee Malzahn
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  3. Yiping Qi
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Corresponding author

Correspondence to Yiping Qi .

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Editors and Affiliations

  1. Department of Agronomy and Horticulture, University of Nebraska – Lincoln, Lincoln, Nebraska, USA

    L. Mark Lagrimini

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Lowder, L.G., Malzahn, A., Qi, Y. (2018). Plant Gene Regulation Using Multiplex CRISPR-dCas9 Artificial Transcription Factors. In: Lagrimini, L. (eds) Maize. Methods in Molecular Biology, vol 1676. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7315-6_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7315-6_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7314-9

  • Online ISBN: 978-1-4939-7315-6

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