Maize pp 197-214 | Cite as

Plant Gene Regulation Using Multiplex CRISPR-dCas9 Artificial Transcription Factors

  • Levi G. Lowder
  • Aimee Malzahn
  • Yiping Qi
Part of the Methods in Molecular Biology book series (MIMB, volume 1676)


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.

Key words

CRISPR-Cas9 Plant transcriptional regulation Artificial transcription factor Transcriptional activator Transcriptional repressor Multiplex Golden Gate assembly Gateway Cloning 



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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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of BiologyEast Carolina UniversityGreenvilleUSA
  2. 2.Department of Plant Science and Landscape ArchitectureUniversity of MarylandCollege ParkUSA

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