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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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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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Simultaneous Gene Excision and Integration by Dual-Guide CRISPR-Cas9.

Michael Spagnuolo1, Mark Blenner2,3

  • 1Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC, USA.

Methods in Molecular Biology (Clifton, N.J.)
|April 13, 2021
PubMed
Summary

This study presents a CRISPR-Cas9 protocol for efficient gene knockout and integration in metabolic engineering. The method combines DNA cutting with two guide RNAs and a donor DNA cassette for precise genetic modifications.

Keywords:
CRISPR-Cas9Gene excisionGenome editingMetabolic engineeringSynthetic biologyYarrowia lipolytica

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Area of Science:

  • Molecular Biology
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • Metabolic engineering often necessitates simultaneous gene knockout and integration.
  • CRISPR-Cas9 is widely used for gene editing but can lead to undesirable mutations or toxic products.
  • Current CRISPR-Cas9 methods for gene integration can be inefficient.

Purpose of the Study:

  • To develop an efficient protocol for combining gene knockout and gene integration using CRISPR-Cas9.
  • To improve the precision and reduce unwanted byproducts in gene editing for metabolic engineering.

Main Methods:

  • Utilized CRISPR-Cas9 with two guide RNAs to create targeted DNA double-strand breaks for gene knockout.
  • Introduced a donor DNA cassette containing the gene for integration at the knockout site.
  • Designed guide RNA target sites flanking homology regions to facilitate both homology-directed repair and homology-mediated end joining.

Main Results:

  • Achieved efficient gene knockout and successful integration of a new gene at the targeted locus.
  • Minimized deletions and undesirable indels, leading to a high proportion of correctly modified genomes.
  • Demonstrated a robust method for combined gene editing operations.

Conclusions:

  • The developed protocol offers a significant advancement for precise gene editing in metabolic engineering.
  • This method enhances the efficiency and reliability of creating complex genetic modifications.
  • It provides a valuable tool for constructing engineered metabolic pathways.