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In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
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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

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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes.

Edward M Barbieri1, Paul Muir1, Benjamin O Akhuetie-Oni1

  • 1Department of Molecular, Cellular, & Developmental Biology, Yale University, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA.

Cell
|November 21, 2017
PubMed
Summary
This summary is machine-generated.

This study introduces a novel multiplex genome engineering method in yeast. It enables precise, efficient DNA modifications without double-strand breaks, creating extensive genetic diversity for pathway engineering.

Keywords:
DNA replicationRad51genome editinghomologous recombinationmetabolic engineeringmultiplex genome engineeringnatural productsssDNA oligodeoxynucleotides

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Genetics

Background:

  • Current genome engineering methods often rely on double-strand breaks and homologous recombination, which can lead to unintended mutations.
  • Multiplex genome engineering is crucial for creating complex genetic diversity efficiently.

Purpose of the Study:

  • To develop a novel, efficient, and precise multiplex genome engineering technology in Saccharomyces cerevisiae.
  • To demonstrate the capability of this technology for combinatorial diversification of biosynthetic pathways.

Main Methods:

  • Utilized annealing of synthetic oligonucleotides at the lagging strand of DNA replication.
  • Bypassed the need for Rad51-directed homologous recombination and double-strand DNA breaks.
  • Achieved simultaneous incorporation of multiple oligonucleotides and targeted mutations.

Main Results:

  • Demonstrated precise chromosome modifications at single base-pair resolution with >40% efficiency.
  • Successfully incorporated up to 12 oligonucleotides and 60 mutations in a single transformation.
  • Generated combinatorial genomic diversity exceeding 10^5 through iterative transformations.
  • Engineered a heterologous β-carotene biosynthetic pathway, yielding variants with altered carotenoid levels due to precise mutations.

Conclusions:

  • The developed method offers a Rad51-independent, double-strand break-free approach for multiplex genome engineering.
  • This technology enables high-efficiency, precise, and combinatorial modification of eukaryotic genomes.
  • The strategy is automatable and applicable for generating significant genomic diversity for various applications, including metabolic engineering.