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Related Concept Videos

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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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Updated: Feb 24, 2026

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation
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Building Cre Knockin Rat Lines Using CRISPR/Cas9.

Yuanwu Ma1, Lianfeng Zhang2, Xingxu Huang3

  • 1Key Laboratory of Human Disease Comparative Medicine, NHFPC, Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, 5 Panjiayuan Nanli, Chaoyang District, Beijing, 100021, China. mayuanwu@cnilas.org.

Methods in Molecular Biology (Clifton, N.J.)
|August 18, 2017
PubMed
Summary

Researchers can now efficiently generate Cre knockin rat lines using the CRISPR/Cas9 genome-editing tool. This streamlined method significantly reduces the time required to create these essential models for studying gene function.

Keywords:
CRISPR/Cas9Conditional knockoutCre/loxPHomologous recombinationRat

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Area of Science:

  • Genetics
  • Molecular Biology
  • Animal Models

Background:

  • Conditional gene inactivation is crucial for studying gene functions during embryogenesis and in adult tissues.
  • The Cre/loxP system is a standard tool for conditional gene manipulation, requiring Cre knockin animal lines for spatial and temporal control.
  • Traditional methods for generating Cre knockin lines are time-consuming and labor-intensive.

Purpose of the Study:

  • To establish a rapid and efficient method for generating Cre knockin rat strains using the CRISPR/Cas9 system.
  • To detail the stepwise procedure for creating precise Cre insertions in rat genomes.
  • To facilitate the generation of valuable genetic tools for biological research.

Main Methods:

  • Utilized the CRISPR/Cas9 genome-editing system for targeted gene modification.
  • Designed and delivered guide RNAs (gRNAs), Cas9 enzyme, and donor DNA templates into fertilized rat eggs.
  • Employed a stepwise protocol including target site selection, RNA preparation, donor construction, pronuclear injection, and genotyping.
  • Focused on direct delivery of CRISPR/Cas9 components and donor DNA for efficient knockin.

Main Results:

  • Successfully generated rat strains carrying Cre genes at targeted loci using CRISPR/Cas9.
  • Demonstrated a stepwise procedure for precise Cre insertion into the rat genome.
  • Achieved the establishment of Cre knockin rat lines within a 6-week timeframe.
  • Validated the efficiency and simplicity of the CRISPR/Cas9 approach compared to traditional methods.

Conclusions:

  • The CRISPR/Cas9 system provides a simple and efficient method for generating Cre knockin rats.
  • This optimized protocol significantly accelerates the creation of essential genetic models for research.
  • The ability to rapidly generate Cre knockin rat lines will advance studies in developmental biology, neuroscience, and other fields.