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High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
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Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression.

Fatwa Adikusuma1,2, Chandran Pfitzner1,3, Paul Quinton Thomas1,3,4

  • 1School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia.

Plos One
|December 7, 2017
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Summary
This summary is machine-generated.

This study introduces novel CRISPR/Cas9 vectors for efficient dual-guide RNA expression. These tools simplify the generation of complex genomic modifications, enhancing CRISPR/Cas9 applications.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • CRISPR/Cas9 technology allows for precise genome editing.
  • Simultaneous expression of two guide RNAs (gRNAs) is crucial for advanced applications like double knock-outs and large deletions.
  • Existing methods for multiplex gRNA expression are often labor-intensive, involving multiple cloning and PCR steps.

Purpose of the Study:

  • To develop a streamlined method for generating customized dual-gRNA expression constructs.
  • To provide researchers with versatile CRISPR/Cas9 vectors for complex genomic modifications.
  • To facilitate the efficient use of CRISPR/Cas9 for advanced gene editing applications.

Main Methods:

  • Development of a series of vectors enabling dual-gRNA expression.
  • Utilized a one-step golden gate cloning reaction with oligonucleotide inserts.
  • Tested vector efficiency via nucleofection in mouse embryonic stem cells.

Main Results:

  • Demonstrated highly efficient target locus cleavage using the developed dual-guide plasmids.
  • Constructs are available as Cas9-nuclease or Cas9-nickase expression vectors.
  • Vectors include options with or without selection markers for flexibility.

Conclusions:

  • The novel vectors simplify the creation of dual-gRNA constructs through a one-step cloning process.
  • These vectors significantly enhance the CRISPR/Cas9 toolbox for researchers.
  • The plasmids will be accessible through the Addgene plasmid repository, promoting wider research use.