TurboKnockout-Pro gene editing technology innovatively combines TurboKnockout^® technology independently developed by Cyagen with BAC recombination technology for improved technological capabilities. Due to the limitation of the capacity of DNA cloning vectors, the exogenous gene fragments used to make genetically modified animals are usually less than 30kb, so some important elements that regulate gene activity are inevitably lost. However, BAC can accommodate foreign DNA up to 300kb. Through BAC recombination, longer fragments of genes and regulatory sequences can be introduced, so as to be closer to the expression pattern of endogenous genes and retain a greater degree of genetic humanization.

Compared with traditional ES cell gene targeting technology, TurboKnockout-Pro not only has the advantages of accurate modification and stable integration effect, but also can greatly shorten the project cycle by realizing multi-step BAC recombination at the ES level. Compared with CRISPR/Cas9 technology, TurboKnockout-Pro technology has no off-target effects and no patent disputes, providing freedom to operate. For these reasons, it is a commonly used technology for new drug research and development projects.

TurboKnockout-Pro gene editing technology integrates BAC recombination to achieve large fragment gene knock-in of up to 300kb without off-target effects. This enables the introduction of more complete human genes into mouse cells to generate whole-gene humanized mouse models. The upgrade of technology saves project development costs by reducing the number of gene editing roundsand is free from patent disputes, which together accelerate the development of new drugs. Case Study: Humanized CD3 Mouse Model

The interspecies sequence preservation is relatively low in the extracellular domains of CD3ε (47% homology between human and mouse) at the amino acid level, CD3δ (57% homology between human and mouse), or CD3γ (60% homology between human and mouse). Therefore, therapeutic antibodies specific for human CD3 cannot effectively activate mouse effector T cells. The establishment of an experimental animal model suitable for evaluating human CD3-specific therapy is required. Cyagen has used TurboKnockout-Pro technology to successfully build a fully genetically humanized CD3 mouse model for an immunotherapy development company, helping the development of innovative multispecific antibody drugs. In addition, Cyagen has cooperated with a number of scientific research institutions, enterprises and institutions to build several large fragment humanized mouse models via TurboKnockout-Pro.

The expression of gene products seen in human disease is not always as high as a transgene, but it should still express to an extent, unlike the non-expression seen in a gene knockout. In many cases, the occurrence of genetic diseases is due to the change of protein structure caused by the mutation of single or several nucleotide bases, which is manifested as abnormal protein activation or inhibition. Therefore, constructing precise gene mutation models that are refined to characterize disease has become the best solution for effective preclinical and translational research. When the human gene under study has a high homology with the mouse gene homologous sequence, it can be simulated by directly making a point mutation mouse model. When the sequence homology between human and mouse is low, it is necessary to make humanized mice with mutations to better mimic human disease phenotypes. In the gene therapy research strategy, the causative gene(s) are found by genomics or proteomics, then combined with the humanized point mutation mouse model for verification, and this has led to many breakthrough discoveries and high-impact publications.