i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Traditional mouse model generation involves technically demanding ex vivo zygote handling and embryo transfer.
• Existing methods like microinjection require specialized equipment and expertise, limiting accessibility.
• Efficient generation of genetically engineered mouse models is crucial for biological research.

Purpose of the Study:

• To introduce a simplified and efficient method for generating genetically engineered mouse models.
• To demonstrate the efficacy of CRISPR ribonucleoprotein delivery into early-stage mouse embryos.
• To establish a technique that bypasses the need for ex vivo zygote manipulation and embryo transfer.

Main Methods:

• Developed improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD).
• Utilized in situ electroporation to deliver CRISPR ribonucleoproteins to E0.7 mouse embryos.
• Generated mouse models with single-base changes, deletions, and knock-ins.

Main Results:

• i-GONAD achieved efficiency comparable to traditional microinjection methods.
• The technique avoids technically difficult ex vivo handling and embryo transfer steps.
• i-GONAD-treated females maintained reproductive function, indicating potential for germline applications.

Conclusions:

• i-GONAD offers a robust, accessible, and efficient alternative for generating genetically engineered mouse models.
• The method simplifies CRISPR-based genome editing in mouse embryos, requiring only basic laboratory equipment.
• i-GONAD holds promise for future applications in germline gene therapy and broader genetic research.