Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes

Area of Science:

• * Molecular Biology
• * Genetics
• * Animal Models

Background:

• * Traditional genome editing in mice relies on microinjection or electroporation of CRISPR reagents into zygotes.
• * These methods necessitate ex vivo embryo handling and transfer to recipient mice, requiring specialized skills and increasing animal usage.
• * Existing protocols for Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) and its improved version (i-GONAD) have been published previously.

Purpose of the Study:

• * To provide updated technical insights into the improved-GONAD (i-GONAD) method for genome editing in mice.
• * To consolidate the detailed i-GONAD protocol in one accessible location for researchers.
• * To highlight the advantages of i-GONAD in simplifying genome editing procedures and reducing animal use.

Main Methods:

• * The i-GONAD method involves in situ delivery of CRISPR reagents into the oviduct of anesthetized pregnant mice.
• * Nucleic acids are delivered using a mouthpiece-controlled glass micropipette under a dissecting microscope.
• * Electroporation of the oviduct facilitates reagent entry into zygotes, followed by natural gestation and delivery.

Main Results:

• * The i-GONAD procedure bypasses the need for ex vivo embryo manipulation and transfer.
• * This method eliminates the requirement for pseudopregnant recipient mice.
• * The i-GONAD approach significantly reduces the overall number of animals required for genome editing experiments.

Conclusions:

• * The i-GONAD method offers a streamlined and more ethical approach to generating genome-edited mice.
• * This technique simplifies the process, making genome editing more accessible.
• * Further adoption of i-GONAD can contribute to a reduction in animal testing for genetic research.