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Updated: Jan 19, 2026

DNA Replication: Semiconservative, 5'-3' DNA Synthesis
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Nuclease dead Cas9 is a programmable roadblock for DNA replication.

Kelsey S Whinn1,2, Gurleen Kaur1,2, Jacob S Lewis1,2

  • 1School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, New South Wales, 2522, Australia.

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|September 19, 2019
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Summary

Researchers developed a novel DNA roadblock using inactivated Cas9 (dCas9) to study molecular machine interactions. This tool effectively halts DNA replication forks from various organisms in vitro, enabling new mechanistic studies.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Studying interactions between DNA-bound molecular machines is challenging due to limited experimental tools.
  • Understanding how these machines collide and affect DNA processes is crucial for molecular biology.

Purpose of the Study:

  • To develop a novel, targetable protein-DNA roadblock for in vitro studies.
  • To investigate the mechanisms of enzymatic activities on DNA substrates when encountering obstacles.

Main Methods:

  • Repurposing a catalytically inactivated Cas9 (dCas9) construct as a protein-DNA roadblock.
  • Utilizing a dCas9-guideRNA complex for specific DNA targeting.
  • Demonstrating replication fork arrest in vitro using the dCas9 roadblock.

Main Results:

  • The dCas9-guideRNA complex successfully functions as a generic and targetable DNA roadblock.
  • Replication forks from viral, bacterial, and eukaryotic sources were arrested in vitro by the dCas9 roadblock.
  • The study validates the utility of dCas9 as a tool for studying DNA-associated processes.

Conclusions:

  • The inactivated Cas9 (dCas9) serves as a versatile tool for creating targeted DNA roadblocks.
  • This method facilitates the study of molecular machine collisions and their impact on DNA replication.
  • The developed dCas9 roadblock system offers a new avenue for in vitro mechanistic investigations in molecular biology.