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Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

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Negative selection and stringency modulation in phage-assisted continuous evolution.

Jacob C Carlson¹, Ahmed H Badran¹, Drago A Guggiana-Nilo²

¹1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

Nature Chemical Biology

|February 4, 2014

Summary

This summary is machine-generated.

Phage-assisted continuous evolution (PACE) accelerates biomolecule engineering. New PACE methods enable evolving radically altered or highly specific activities, demonstrated by evolving T7 RNA polymerase with greatly enhanced substrate specificity.

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

Molecular Biology
Biotechnology
Biochemistry

Background:

Phage-assisted continuous evolution (PACE) is a powerful technique for accelerating laboratory evolution experiments.
Existing PACE methods have limitations in evolving biomolecules with novel or highly specific activities.

Purpose of the Study:

To enhance the scope and capabilities of the PACE method.
To enable the evolution of biomolecules with radically altered or highly specific new activities.
To demonstrate these advancements by evolving T7 RNA polymerase.

Main Methods:

Implementation of small molecule-controlled modulation of selection stringency.
Development of a general negative selection for counterselection against undesired activities.
Integration of these developments into a single PACE experiment.

Main Results:

Successfully evolved mutant T7 RNA polymerase enzymes with approximately 10,000-fold altered substrate specificities.
Achieved evolution of specific activities directly from inactive starting libraries.
Evolved enzymes exhibited target substrate specificity exceeding wild-type RNA polymerases while maintaining wild-type-like activity.

Conclusions:

The enhanced PACE method significantly expands the capabilities for directed evolution of biomolecules.
These advancements allow for the direct evolution of novel and highly specific biomolecular functions.
The evolved T7 RNA polymerase variants represent a significant advancement in enzyme engineering.