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Updated: Feb 8, 2026

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9.

Tadas Jakočiūnas1, Lasse E Pedersen1, Alicia V Lis1

  • 1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.

Metabolic Engineering
|July 9, 2018
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Summary
This summary is machine-generated.

This study introduces a robust directed evolution method using CRISPR technology for efficient large-scale mutagenesis in genomic contexts. The approach enhances enzyme production, demonstrating its power for metabolic engineering and synthetic biology applications.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Directed evolution is crucial for protein engineering.
  • Existing methods often face limitations in scale and genomic integration.
  • CRISPR technology offers precise genome editing capabilities.

Purpose of the Study:

  • To develop a robust method for directed evolution using large-scale mutagenesis within genomic contexts.
  • To enhance the production of metabolic gene products through engineered enzymes.
  • To leverage CRISPR-Cas9 for efficient integration of mutant libraries.

Main Methods:

  • Utilized error-prone PCR to generate mutant libraries.
  • Employed Cas9-mediated genome integration for inserting large donor variants into Saccharomyces cerevisiae.
  • Analyzed mutation frequency and distribution in integrated genomic loci.

Main Results:

  • Achieved high efficiencies (98-99%) for Cas9-mediated genome integration.
  • Demonstrated even mutation frequency distribution across integrated fragments.
  • Engineered mevalonate pathway enzymes in yeast, resulting in up to 11-fold higher isoprenoid production.

Conclusions:

  • The developed method enables efficient, large-scale mutagenesis in native genomic environments.
  • This approach significantly enhances the directed evolution of metabolic enzymes.
  • The method expands CRISPR applications for protein engineering and synthetic biology.