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Programmable DNA looping using engineered bivalent dCas9 complexes.

Nan Hao1, Keith E Shearwin2, Ian B Dodd2

  • 1Department of Molecular and Cellular Biology, School of Biological Sciences, The University of Adelaide, Adelaide, SA, 5005, Australia. nan.hao@adelaide.edu.au.

Nature Communications
|November 22, 2017
PubMed
Summary
This summary is machine-generated.

Scientists created new CRISPR-based tools to program DNA loops, which are crucial for gene regulation. These novel reagents enable precise manipulation of DNA looping, advancing genetic engineering and our understanding of gene control.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • DNA looping is essential for gene regulation but tools for its manipulation are limited.
  • Understanding and controlling DNA looping is key to advancing genetic engineering.

Purpose of the Study:

  • To develop novel CRISPR-based reagents for programmable DNA looping.
  • To enable precise manipulation and study of DNA looping in various cell types.

Main Methods:

  • Development of bivalent complexes using cleavage-defective Cas9 proteins linked via heterodimerization or fusion.
  • Optimization of reagents using computational modeling.
  • Validation through quantitative DNA looping assays in E. coli.
  • Application in activating endogenous genes by rewiring chromosomal DNA.

Main Results:

  • Successful creation of CRISPR-based DNA looping reagents.
  • Demonstrated looping efficiency of approximately 15% for a 4.7 kb loop, with improvements via multiplexing.
  • Activation of endogenous norVW genes by targeted DNA rewiring.
  • Proof-of-concept for manipulating gene regulation through engineered DNA loops.

Conclusions:

  • CRISPR-based reagents offer a powerful new method for creating programmable DNA loops.
  • These tools can be used to study endogenous DNA loops and engineer novel regulatory circuits.
  • The technology has broad potential applications in diverse cell types for genetic research and biotechnology.