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Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically modified organisms are called transgenic organisms.
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Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants
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A multiplex guide RNA expression system and its efficacy for plant genome engineering.

Youngbin Oh1, Bora Lee1, Hyeonjin Kim1

  • 1Department of Biological Sciences, KAIST, Daejeon, 34141 Republic of Korea.

Plant Methods
|March 20, 2020
PubMed
Summary

A new PCR-free cloning system enables high-throughput plant genome editing by expressing multiple guide RNAs (gRNAs) simultaneously. This method enhances editing efficiency and facilitates large deletions between targeted sites.

Keywords:
CRISPR-Cas9Golden gate assemblyMultiplex gRNAsPlant genome editing

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genome Engineering

Background:

  • The Streptococcus pyogenes CRISPR system utilizes Cas9 endonuclease (SpCas9) and guide RNA (gRNA) for targeted DNA cleavage.
  • SpCas9's potential for genome editing is theoretically limited only by the number of gRNAs that can bind to specific genomic loci.

Purpose of the Study:

  • To develop a PCR-free, multiplex gRNA cloning system for efficient plant genome editing.
  • To validate the efficacy of this system in wild tobacco (Nicotiana attenuata).

Main Methods:

  • A two-step cloning process involving single-stranded oligonucleotides and a pGRNA vector.
  • Assembly of tRNA-gRNA units using Golden Gate assembly with a SpCas9 expression cassette.
  • Validation through targeted deep sequencing in protoplasts and transformed plants.

Main Results:

  • Successful implementation of a PCR-free multiplex gRNA cloning system for plant genome editing.
  • Demonstrated increased editing efficiency and induction of large deletions between two proximal SpCas9-gRNA cleavage sites.
  • Validated system performance in Nicotiana attenuata protoplasts and whole plants.

Conclusions:

  • The developed multiplex gRNA expression system allows for high-throughput generation of single binary vectors.
  • This system significantly enhances the efficiency of plant genome editing applications.