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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

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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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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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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
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Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse.

Kyriel M Pineault1,2, Ana Novoa2, Anastasiia Lozovska2

  • 1Department of Cell and Regenerative Biology, University of Wisconsin-Madison, USA.

Methodsx
|November 1, 2019
PubMed
Summary

Researchers developed two CRISPR/Cas9 methods to efficiently generate large, complex gene edits in mouse models. These advanced techniques improve genome modification for disease research and gene function studies.

Keywords:
CRISPR/Cas9CRISPR/Cas9-mediated large genetic modifications in mouseGene-editingMicroinjectionMouseStreptavidin/biotinZygotessDNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Genetically modified model organisms are crucial for biological research.
  • CRISPR/Cas9 technology has accelerated small gene editing but struggles with large modifications.
  • Homology-directed repair efficiency is key for precise large-scale genome editing.

Purpose of the Study:

  • To present novel CRISPR/Cas9 methods for generating large and complex gene edits in mice.
  • To improve the efficiency of homology-directed repair for sophisticated genetic modifications.
  • To overcome limitations in current CRISPR/Cas9 techniques for generating advanced model organisms.

Main Methods:

  • Developed an alternative protocol for preparing long single-stranded DNA (lssDNA) templates.
  • Utilized a dual single-guide RNA (sgRNA) strategy with Cas9 and lssDNA.
  • Employed a tethering approach using biotinylated double-stranded DNA (dsDNA) templates and a Cas9-streptavidin fusion protein.

Main Results:

  • Successfully generated large and complex gene edits in mouse models using the described CRISPR/Cas9 methods.
  • Demonstrated the feasibility of using two sgRNAs with Cas9 and lssDNA for significant genome modification.
  • Showcased the effectiveness of the Cas9-streptavidin/biotinylated-dsDNA tethering system for precise large DNA modifications.

Conclusions:

  • The presented CRISPR/Cas9 methods offer efficient solutions for creating complex genetic modifications in model organisms.
  • These advancements facilitate the generation of sophisticated models for studying gene function and human diseases.
  • The study highlights innovative strategies to enhance homology-directed repair for large-scale genome engineering.