A research group led by Professor Hiromitsu Nakauchi of the University of Tokyo has developed a method for easily performing genetic recombination by genome editing using viral infection of fertilized mammalian eggs.It is expected that it will be possible to create genetically modified animals and treat complex genetic diseases without special techniques.

 Gene insertion by genome editing technology is the basis of basic research using genetically modified animals, and is also a useful technology for the treatment of genetic diseases.Recent technological advances have made it possible to apply genome editing technology at the fertilized egg stage, but unlike gene disruption, gene insertion requires advanced techniques and limits the length of genes that can be introduced. ..As a highly efficient and simple method for introducing genes into cells, there is a method using a viral vector, but unlike general cells, fertilized mammalian eggs have the zona pellucida (the sugar protein that surrounds the fertilized eggs before implantation in the uterus). It has been difficult to apply because it is protected by the membrane.

 The research group found that the single-stranded DNA virus "adeno-associated virus" can infect various fertilized mammalian eggs through the zona pellucida, unlike common viral vectors.Utilizing this characteristic, special equipment and technology are used by combining the gene modification technology "CRISPR / Cas9 genome editing" by electroporation, which punches holes in the cell membrane with high voltage, with the introduction of donor DNA using an adeno-associated virus vector. We succeeded in knocking in a long-chain foreign gene at the fertilized egg stage without using.Furthermore, using the same method, we succeeded in gene therapy of nude mice, which are immunodeficiency model animals.

 This method is considered to be applicable to a wide range of mammals, and is expected to be a basic technology in biological and medical research, and to enable gene therapy in domestic animals with genetic diseases.

Paper information:[IScience] Intra-embryo gene cassette knock-in by CRISPR / Cas9-mediated genome editing with adeno-associated viral vector

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