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Related Concept Videos

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Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Related Experiment Video

Updated: Jul 1, 2025

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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Positive Selection Screens for Programmable Endonuclease Activity Using I-SceI.

Michael A Mechikoff1, Kok Zhi Lee2, Kevin V Solomon3

  • 1Department of Biology, US Air Force Academy, Colorado Springs, CO, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 12, 2024
PubMed
Summary
This summary is machine-generated.

We developed a novel I-SceI-based platform for flexible screening of DNA endonucleases. This method enriches for more powerful enzyme variants, overcoming limitations of stringent selection methods.

Keywords:
CRISPRDirected evolutionEnrichmentI-SceIPositive selectionEndonuclease

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • High-throughput screening is crucial for enzyme discovery and engineering.
  • Existing positive selection screens can be overly stringent, leading to high false-negative rates.
  • Novel programmable DNA endonucleases require flexible and less restrictive screening platforms.

Purpose of the Study:

  • To develop a novel I-SceI-based platform for screening programmable DNA endonucleases.
  • To create a more flexible and less restrictive selection system compared to existing methods.
  • To enable efficient characterization and enrichment of active endonuclease variants.

Main Methods:

  • Developed an I-SceI-based system in E. coli where genome cleavage inhibits growth.
  • Utilized activity-dependent growth rescue via plasmid curing or cleavage of the I-SceI expression plasmid by candidate endonucleases.
  • Demonstrated the platform with Cas9, adapting it for various programmable DNA endonucleases.

Main Results:

  • The platform allows for activity-dependent enrichment of endonuclease candidates.
  • More active variants preferentially proliferate, leading to successful enrichment.
  • The system effectively characterizes single candidates and enriches variants from libraries.

Conclusions:

  • The novel I-SceI-based platform offers a flexible and powerful tool for screening DNA endonucleases.
  • This system overcomes limitations of stringent selection, reducing false negatives.
  • The protocol is adaptable for diverse endonucleases and applications like biodiversity mining and directed evolution.