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Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems

  • Levi G. Lowder
  • Joseph W. PaulIII
  • Yiping Qi
Part of the Methods in Molecular Biology book series (MIMB, volume 1629)

Abstract

Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). These tools are especially useful for studying gene function and interaction within various regulatory networks. First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX). When multiple chimeric dCas9-effector fusions are guided to gene regulatory regions via CRISPR gRNAs, they can modulate expression of transcript levels in planta. The protocol presented here provides a detailed procedure for activating AtPAP1 and repressing AtCSTF64 in Arabidopsis thaliana. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.

Key words

CRISPR-Cas9 Transcriptional regulation Artificial transcription factors Transcriptional activation Transcriptional repression Multiplex CRISPR-Cas9 Multiplex gRNA cloning 

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Levi G. Lowder
    • 1
  • Joseph W. PaulIII
    • 1
  • Yiping Qi
    • 1
  1. 1.Department of BiologyEast Carolina UniversityGreenvilleUSA

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