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

Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos' genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.