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In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Related Experiment Video

Updated: Jul 24, 2025

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

Published on: December 16, 2022

An efficient evaluation system for factors affecting the genome editing efficiency in mouse.

Yusuke Sakai¹, Yuri Okabe¹, Gen Itai^2,3

¹Institute for Disease Modeling, Kurume University School of Medicine, 67 Asahimachi, Kurume city, Fukuoka 830-0011, Japan.

Experimental Animals

|July 5, 2023

Summary

Researchers developed a new genome editing system to improve precise gene knock-in efficiency in mice. This system successfully identified a compound that enhances knock-in rates in mouse embryos, aiding genetic disease model creation.

Keywords:

CRISPR-Cas9 genome editing knock-in restriction fragment length polymorphism (RFLP)

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Last Updated: Jul 24, 2025

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

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Published on: April 2, 2020

Area of Science:

Laboratory animal science
Molecular biology
Genetics

Background:

Genome editing is crucial for creating genetic disease models and studying gene function in laboratory animals.
Precise gene knock-in via homologous recombination is less efficient than gene knockout via non-homologous end joining, posing a significant challenge.
Existing solutions for improving knock-in efficiency are insufficient.

Purpose of the Study:

To establish a robust system for evaluating genome editing efficiency at the mouse Tyr gene locus.
To identify small-molecule compounds that enhance precise gene knock-in efficiency in mouse embryos.
To facilitate the screening and development of novel genome editing strategies.

Main Methods:

Developed a genome editing system utilizing Cas9 and a specific donor template targeting the mouse Tyr gene locus.
Designed primers and utilized restriction fragment length polymorphism (RFLP) for easy genotyping of wild-type, knockout, and knock-in alleles.
Incorporated the H420R mutation in tyrosinase to enable visual identification of knock-in mice via coat color.
Screened various small-molecule compounds for their effect on knock-in efficiency in mouse embryos.

Main Results:

The established system allows for straightforward in vitro and in vivo genotyping of genome-edited mouse embryos.
Wild-type, knockout, and knock-in genotypes were distinguishable using RFLP analysis of PCR products.
The H420R mutation facilitated the identification of knock-in mice with distinct coat color patterns.
A specific small-molecule compound was identified that significantly improved the knock-in efficiency in mouse embryos.

Conclusions:

The developed genome editing system is effective for assessing gene editing outcomes at the Tyr locus.
This system is highly suitable for high-throughput screening of compounds aimed at enhancing precise gene knock-in efficiency.
The findings contribute to advancing genome editing technologies for creating sophisticated genetic models.