In terms of drug development, currently, the most frequently used gene editing technology is the preparation of animal models.
Since the homology of the immune checkpoint gene of the common mouse to the corresponding human gene is only about 60%, an antibody that acts on a human protein generally does not recognize a protein in a mouse. Therefore, it is impossible to use ordinary mice for the evaluation of the drug efficacy of antibody drugs, and it is necessary to study a targeted drug by using a certain technique to convert a mouse gene as a gene target into a homologous gene of an adult, that is, a humanized mouse, which makes mice express human proteins or antibodies for the development of new therapeutics and drugs.
At present, animal model gene editing technology can be divided into two categories: ES cell targeting technology and CRISPR/Cas9 technology.
In which, ES targeting is to carry out DNA homologous recombination in mouse embryonic stem cells (ES cells), re-injecting ES cells into the blastocyst cavity to form chimeric embryos, and developing chimeric mice in pseudopregnant mice. The chimeric mouse is then mated with wild mice to transfer genetic information in the ES cells to the offspring mice, which is featured of accurate and basically free of off-target, this method can carry out various complex genetic modification, however, the disadvantage is inefficient, time-consuming, laborious, costly, and only applicable to mice. In recent years, there have also been attempts to improve the company. The current improved version of the ES target, TurboKnockout technology, has shortened the build cycle from one year to half a year and almost equal to CRISPR, and the cost has also been reduced.
At present, the most popular and widely used CRISPR/Cas9 system is composed of Cas9 protein and guide RNA. Cas9 contains two active sites, RuvC at the amino terminus and HNH at the middle of the protein, which play a role in crRNA maturation and double-strand DNA splicing, causing DNA double-strand breaks. When the DNA is broken and a DNA fragment homologous to the damaged DNA is present in the nucleus, the foreign DNA fragment can be introduced at the destination site by homologous-mediated double-stranded DNA repair, thereby achieving the effect of fragment knocking or editing. The advantages are efficient, fast, simple, inexpensive, and can be used in different species; the disadvantage is that there is always an unpredictable and uncontrollable off-target risk, and it is not suitable for complex genetic modification projects.
For CRISPR technology, some researchers have pointed out that the current off-target problem can not be completely avoided. ‘Although off-target prediction and a certain degree of prevention can be performed through genome-wide off-target risk calculations, the off-target issue still needs to be solved for scientific research with very high rigor.’
The patented CRISPR technology requires an authorization fee, while ES targeting is relatively high in both time and cost. Therefore, how to obtain high-quality animal model services at a lower cost has become a concern of many research institutions.
It is a valuable development direction to build a resource library by professional gene knockout model animal commercial technology platform or related machine. The continuously optimized cryopreservation technology can further improve the recovery rate and birth rate of sperm and embryos of animals, and has ideal stability, which provides stable technical support for the construction of gene knockout mouse resource pool.
For researchers, the same strains, the cost of custom animal models is also significantly higher than the purchase of commercial institutions, especially in the study of Me-too, Me-better drugs.
It is foreseeable that with the output of more high-quality animal models, the pace of accelerated biomedical research will be more stable and bring more new biopharmaceuticals to the world.