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Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement.

Yaya Hao1, Jiang Li2,3, Qian Li1

  • 1School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules, Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200240, China.

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Summary

Researchers developed a programmable CRISPR-Cas9 system using toehold switches to control single guide RNA (sgRNA) conformation. This innovation enables precise genome engineering and imaging in living cells.

Keywords:
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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Genome Engineering

Background:

  • CRISPR-Cas9 technology has revolutionized genome engineering but requires enhanced precision in targeting.
  • Controlling Cas9 activity dynamically within living cells remains a challenge for advanced applications.

Purpose of the Study:

  • To engineer a novel system for programmable activation of CRISPR-Cas9.
  • To develop a method for precise control over single guide RNA (sgRNA) conformation.
  • To enable multiple, orthogonal responses for advanced genome imaging and engineering.

Main Methods:

  • Utilized a toehold-switch-based mechanism to modulate sgRNA conformation.
  • Implemented strand displacement for input-responsive regulation of Cas9 activity.
  • Demonstrated orthogonal activation and suppression using distinct DNA inputs.

Main Results:

  • Successfully engineered a programmable CRISPR-Cas9 activation circuit responsive to multiple inputs.
  • Achieved orthogonal control over Cas9 targeting through engineered sgRNA conformations.
  • Showcased simultaneous and orthogonal responses for multicolor display of multiple genome loci.

Conclusions:

  • The toehold-switch approach offers a new strategy for programming CRISPR-Cas9 activity in living cells.
  • This method enhances control over genome engineering and facilitates advanced applications like live-cell genome imaging.