CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CRISPR/Cas9 facilitates rapid gene knockout animal generation.
• Large fragment knock-in into embryos is challenging without microinjection.
• Viral vectors are typically unsuitable for pre-implantation embryos due to the zona pellucida.

Purpose of the Study:

• To investigate adeno-associated virus (AAV) as a vector for large fragment knock-in into mammalian zygotes.
• To develop a microinjection-free method for gene editing in early embryos.

Main Methods:

• Electroporation of Cas9 ribonucleoprotein complexes.
• Delivery of donor DNA using adeno-associated virus (AAV) vectors.
• Infection of mammalian zygotes through the intact zona pellucida.

Main Results:

• AAV successfully infected zygotes of various mammals, penetrating the zona pellucida.
• AAV-mediated donor DNA delivery combined with Cas9 ribonucleoprotein electroporation enabled large fragment knock-in.
• This method achieved large fragment knock-in without the need for microinjection.

Conclusions:

• Adeno-associated virus (AAV) is an effective vector for delivering donor DNA for large fragment knock-in into mammalian embryos.
• This technique offers a novel, microinjection-free approach for genome editing in early embryonic development.
• The findings pave the way for more efficient generation of genetically modified animals.