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Continuous Hypermutation and Evolution of Luciferase Variants.

Tanya Hadjian1, Ria A Deshpande2,3,4, Zachary R Torrey1

  • 1Department of Chemistry, University of California, Irvine, California 92697, United States.

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|December 15, 2025
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Summary
This summary is machine-generated.

This study introduces an orthogonal DNA replication (OrthoRep) system for continuous hypermutation, streamlining luciferase engineering. This novel method rapidly evolves luciferases for improved bioluminescent reporters without manual library generation.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Luciferases are crucial tools for bioimaging and biosensing.
  • Current luciferase optimization methods are inefficient and labor-intensive, involving manual screening of mutant libraries.
  • There is a need for streamlined and automated approaches to accelerate enzyme engineering.

Purpose of the Study:

  • To develop a streamlined platform for luciferase engineering using continuous hypermutation.
  • To eliminate the need for repetitive manual library generation in enzyme evolution.
  • To engineer novel luciferase variants with enhanced performance.

Main Methods:

  • Implementation of an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of the GeNL luciferase.
  • Short cycles of culturing and screening to evolve the enzyme.
  • Characterization of novel luciferase variants in cell models.

Main Results:

  • Successful evolution of GeNL luciferase variants using the OrthoRep system.
  • Identification of new variants exhibiting improved light output with an inexpensive, noncognate luciferin.
  • Demonstration of the engineered luciferases' utility in cell-based assays.

Conclusions:

  • The OrthoRep system and continuous hypermutation provide a viable and efficient method for luciferase engineering.
  • This approach significantly accelerates the development of bioluminescent reporters.
  • The engineered luciferases show promise for advanced bioimaging and biosensing applications.