Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

16.7K
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.
16.7K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Distal enhancer-insulator module of GDF6 is essential for cochlear formation.

JCI insight·2026
Same author

Hepatocyte-Specific Deletion of Betaine-Homocysteine Methyltransferase Disrupts Methionine Metabolism and Promotes the Spontaneous Development of Hepatic Steatosis.

Biomolecules·2026
Same author

Inflammatory macrophage-derived plasminogen activator inhibitor-1 exacerbates inflammation through efferocytosis inhibition.

Cell death discovery·2026
Same author

Ribonuclease 4 Functions in Nociceptor-Mediated Nerve Homeostasis.

Nature communications·2026
Same author

Reduced <i>Akr1b7</i> signaling drives ovarian aging and reproductive dysfunction.

iScience·2026
Same author

A single microRNA miR-195 rescues the arrested B cell development induced by EBF1 deficiency.

eLife·2026

Related Experiment Video

Updated: Feb 16, 2026

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
08:48

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

Published on: June 24, 2022

5.6K

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

Hiromi Miura1,2, Rolen M Quadros3, Channabasavaiah B Gurumurthy3,4

  • 1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.

Nature Protocols
|December 22, 2017
PubMed
Summary

Efficient Additions with single-stranded DNA inserts-CRISPR (Easi-CRISPR) technology significantly improves gene knock-in and replacement in mouse models. This method uses single-stranded DNA donors for high-efficiency genome editing, reducing model generation time.

More Related Videos

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes
07:17

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

Published on: December 16, 2022

4.2K
Generation of Genetically Modified Mice through the Microinjection of Oocytes
10:19

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Published on: June 15, 2017

21.8K

Related Experiment Videos

Last Updated: Feb 16, 2026

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
08:48

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

Published on: June 24, 2022

5.6K
Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes
07:17

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

Published on: December 16, 2022

4.2K
Generation of Genetically Modified Mice through the Microinjection of Oocytes
10:19

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Published on: June 15, 2017

21.8K

Area of Science:

  • Molecular Biology
  • Genetics
  • Bioengineering

Background:

  • CRISPR/Cas9 genome editing is effective for gene knockouts but inefficient for knock-in or replacement.
  • Existing methods using double-stranded DNA donors yield low efficiency (1-10%) for complex mouse model generation.
  • Most research mouse models require knock-in or gene replacement strategies.

Purpose of the Study:

  • To develop a highly efficient method for generating knock-in and gene replacement mouse models.
  • To overcome the limitations of current CRISPR/Cas9-based genome editing techniques for complex genetic modifications.
  • To establish a reliable and faster protocol for creating genetically engineered mouse models.

Main Methods:

  • Utilized long single-stranded DNAs (ssDNAs) as donors for CRISPR/Cas9-mediated genome editing.
  • Applied the Easi-CRISPR (Efficient Additions with ssDNA inserts-CRISPR) method for both gene insertion and replacement.
  • Optimized the protocol for generating founder mice within approximately two months.

Main Results:

  • Achieved significantly higher efficiency for knock-in and gene replacement compared to previous methods.
  • Demonstrated typical efficiencies ranging from 30-60%, with some cases reaching up to 100%.
  • Successfully generated founder mice using the Easi-CRISPR protocol in approximately two months.

Conclusions:

  • Easi-CRISPR, utilizing ssDNA donors, provides a highly efficient and rapid approach for generating complex mouse models.
  • This technology overcomes previous limitations in CRISPR/Cas9-mediated knock-in and gene replacement.
  • The Easi-CRISPR method is suitable for routine use in creating genetically engineered mouse models for research.