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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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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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Homologous Recombination02:31

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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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Conservative Site-specific Recombination and Phase Variation02:53

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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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Updated: Nov 9, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Gene Excision 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

CRISPR-Cas9 gene editing creates DNA breaks repaired by non-homologous end joining, often resulting in indels. Using two guide RNAs enables precise gene excision by creating simultaneous DNA breaks.

Keywords:
CRISPR-Cas9Gene excisionGenome editingMetabolic engineeringSynthetic biologyYarrowia lipolytica

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 technology is widely used for gene editing.
  • Non-homologous end joining (NHEJ) is a common DNA repair pathway following CRISPR-Cas9 induced double-strand breaks.
  • Indels generated by NHEJ can revert to wild-type sequences, complicating analysis.

Purpose of the Study:

  • To explore an alternative CRISPR-Cas9 application for precise gene modification.
  • To investigate the outcomes of dual guide RNA-mediated DNA cleavage.
  • To develop methods for reliable gene excision rather than simple indels.

Main Methods:

  • Utilizing CRISPR-Cas9 with two distinct guide RNAs targeting adjacent sites.
  • Analyzing DNA repair outcomes following dual-site cleavage.
  • Employing sequencing and other assays to characterize genetic modifications.

Main Results:

  • Simultaneous cutting at two target sites can occur.
  • Repair of dual-site breaks can lead to the precise excision of the intervening DNA sequence.
  • Single indels may form at individual cut sites, while gene excision occurs with dual-site repair.

Conclusions:

  • Dual guide RNA CRISPR-Cas9 strategies offer a method for targeted gene excision.
  • This approach provides a more defined genetic modification compared to single-site indel formation.
  • Precise gene excision via CRISPR-Cas9 can be achieved by leveraging dual-site targeting and repair mechanisms.