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Related Experiment Video

Updated: Jul 17, 2025

Author Spotlight: Advancing Protein Engineering – Harnessing Evolution Through PRANCE and Lab Automation

Author Spotlight: Advancing Protein Engineering – Harnessing Evolution Through PRANCE and Lab Automation

Published on: January 12, 2024

Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

Jordan L Doman¹, Smriti Pandey¹, Monica E Neugebauer¹

¹Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.

|September 1, 2023

Summary

This summary is machine-generated.

Researchers engineered smaller and more efficient prime editing tools for precise genome editing. These advanced prime editors show improved therapeutic editing in cells and enable longer DNA insertions in vivo.

Keywords:

CRISPR-Cas9 directed evolution genome editing guide RNAs pegRNAs phage-assisted continuous evolution prime editing protein engineering

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Area of Science:

Molecular Biology
Genome Engineering
Protein Engineering

Background:

Prime editing is a versatile technology for precise genome modification in living cells.
Current prime editors face limitations in size and efficiency, impacting their therapeutic applications.

Purpose of the Study:

To develop smaller and more efficient prime editing systems through protein evolution and engineering.
To enhance the performance of prime editors for therapeutic applications and in vivo gene editing.

Main Methods:

Utilized phage-assisted evolution to improve reverse transcriptase efficiency.
Engineered novel Cas9 domains to enhance prime editing capabilities.
Tested prime editor variants in patient-derived fibroblasts and primary human T-cells.
Evaluated in vivo editing efficiency using dual-AAV delivery in murine brain models.

Main Results:

Achieved up to 22-fold improvement in editing efficiencies for compact reverse transcriptases.
Developed prime editors 516-810 base pairs smaller than PEmax.
Identified reverse transcriptase specialization for different edit types.
Demonstrated enhanced therapeutic editing in patient cells and T-cells.
Enabled 40% loxP insertion in the murine brain cortex, a 24-fold improvement for in vivo long insertions.

Conclusions:

Engineered prime editors (PE6 variants) exhibit significantly improved efficiency and reduced size.
These advancements broaden the potential of prime editing for therapeutic applications and in vivo genome engineering.
The developed tools offer enhanced capabilities for precise and efficient gene modification in various cell types and organisms.