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Related Experiment Videos

In Planta Processing of the SpCas9-gRNA Complex.

Masafumi Mikami1,2, Seiichi Toki1,2,3, Masaki Endo2

  • 1Graduate School of Nanobioscience, Yokohama City University, 22- 2 Seto, Yokohama, Kanagawa 236-0027, Japan.

Plant & Cell Physiology
|October 18, 2017
PubMed
Summary

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This study introduces a novel CRISPR/Cas9 system for plant genome editing. It efficiently produces functional guide RNA (gRNA) from a single RNA polymerase II transcript using endogenous plant systems, achieving high mutagenesis rates in rice.

Area of Science:

  • Plant Biotechnology
  • Genome Engineering
  • Molecular Biology

Background:

  • CRISPR/Cas9 genome editing in plants typically uses separate promoters for Cas9 protein (RNA polymerase II) and guide RNA (pol III).
  • Limited characterization of plant pol III promoters hinders optimal gRNA expression.
  • Existing methods for processing pol II transcripts into functional gRNAs often require specific RNA processing systems like ribozymes or Csy4.

Purpose of the Study:

  • To develop a simplified CRISPR/Cas9 system for plant genome editing.
  • To demonstrate efficient functional guide RNA processing from a single pol II transcript using endogenous plant RNA processing.
  • To evaluate the efficiency of this novel system in targeted mutagenesis in rice.

Main Methods:

  • Co-transcription of SpCas9 mRNA and gRNA from a single pol II promoter as a single RNA molecule.
Keywords:
CRISPR/Cas9RibozymeTargeted mutagenesis

Related Experiment Videos

  • Binding of translated SpCas9 protein to the co-transcribed RNA.
  • Processing of the RNA complex by endogenous plant RNA cleavage systems to generate functional SpCas9-gRNA complexes.
  • Application of the system for targeted mutagenesis in rice.

    • Main Results:

    • Functional SpCas9-gRNA complexes were efficiently formed without external processing systems.
    • The novel fused system achieved targeted mutagenesis efficiency comparable to existing methods.
    • Up to 100% mutagenesis efficiency was observed in rice using the developed system.

    Conclusions:

    • A novel, simplified CRISPR/Cas9 system utilizing endogenous plant RNA processing for gRNA generation has been developed.
    • This system offers an efficient alternative for stable SpCas9-gRNA expression in plants.
    • The findings have implications for improving genome editing technologies, including RNA virus vector-mediated systems.