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nCas9-based method for rolling-circle DNA substrate generation.

Nischal Sharma1, Kelsey S Whinn1, Harshad Ghodke1

  • 1Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, 2522, Australia.

Analytical Biochemistry
|April 27, 2025
PubMed
Summary

We developed a new method using nickase Cas9 (nCas9) to create customized rolling-circle DNA templates for enhanced DNA replication. This approach offers precise control over replication fork topology and gap size, improving in vitro studies.

Keywords:
Guide RNAsNickase Cas9Nucleic-acid biochemistryReplicationRolling-circle DNASingle-molecule imaging

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Area of Science:

  • Molecular Biology
  • Biotechnology

Background:

  • Rolling-circle DNA replication is crucial for diagnostics, sequencing, and nanotechnology.
  • Current methods for creating rolling-circle DNA templates have limitations in controlling replication fork topology and position.

Purpose of the Study:

  • To develop a straightforward, customizable, and efficient strategy for producing rolling-circle DNA substrates.
  • To enable precise control over replication fork topology and gap size in DNA substrates.

Main Methods:

  • Utilized nickase Cas9 (nCas9) programmed with guide RNAs to target specific DNA sequences.
  • Generated 8-11 bp fragments on an 18-kb plasmid in a one-pot reaction.
  • Ligated a flap oligo to construct a fork with controlled flap length and gap size.

Main Results:

  • Successfully created rolling-circle DNA substrates with precisely controlled flap length and gap size.
  • Demonstrated the application of these substrates in an in vitro single-molecule rolling-circle DNA-replication assay.
  • Showcased the conversion of any plasmid DNA into a rolling-circle template.

Conclusions:

  • The nCas9-based method provides a versatile and efficient way to generate customized rolling-circle DNA templates.
  • This technique allows for the creation of more physiologically relevant DNA templates for research and applications.
  • The developed strategy overcomes limitations of existing methods, offering greater control and flexibility.