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CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.

Changtian Pan1, Xincheng Wu1, Kasey Markel2

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|June 25, 2021
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Researchers developed CRISPR-Act3.0, a robust CRISPR activation (CRISPRa) system for plants. This system enables simultaneous activation of multiple genes, significantly advancing plant gene editing tools for research and applications.

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Area of Science:

  • Plant biotechnology
  • Molecular biology
  • Gene editing

Background:

  • CRISPR activation (CRISPRa) systems are used in plants but struggle with simultaneous multi-gene activation.
  • Developing efficient CRISPRa tools for plants is crucial for metabolic engineering and trait modification.

Purpose of the Study:

  • To develop a highly robust CRISPRa system for plants capable of simultaneous multi-gene activation.
  • To improve existing CRISPRa systems for enhanced activation efficiency and broader targeting scope.
  • To provide a versatile toolbox for plant gene activation in research and translational applications.

Main Methods:

  • Systematic exploration of effector recruitment strategies and transcription activators using deactivated Streptococcus pyogenes Cas9 (dSpCas9).
  • Development of a tRNA-gR2.0 expression system for multi-gene activation.
  • Adaptation of the CRISPRa strategy to CRISPR-dCas12b and development of a near-PAM-less system using SpRY dCas9 variant.

Main Results:

  • CRISPR-Act3.0 achieved fourfold to sixfold higher activation than existing CRISPRa systems in rice, Arabidopsis, and tomato.
  • Enabled robust activation of up to seven genes for metabolic engineering in rice.
  • Facilitated stable, simultaneous modification of multiple traits in Arabidopsis across generations.
  • Elucidated guide RNA targeting rules for effective transcriptional activation.
  • Developed improved CRISPRa systems targeting T-rich PAMs and near-PAM-less variants.

Conclusions:

  • CRISPR-Act3.0 represents a substantial advancement in plant CRISPRa technology.
  • The developed system offers enhanced potency, broader targeting, and robust multi-gene activation capabilities.
  • This toolbox empowers plant researchers for efficient gene activation in foundational and translational studies.