The Exploration Of Using CRISPR/Cas9 And AttP Tandem Sequence To Mediate The Integration Of Multiple Copies Of Target Genes At ROSA26 Locus In Goats

The use of gene editing technology can achieve the removal of species’ own defective genes and the stable integration and specific expression of foreign genes.The use of this technology in the preparation of transgenic animals not only lays a certain foundation for animal improvement and animal mammary gland bioreactors,but also has important significance for the research of various diseases and the establishment of disease models.Among gene editing technologies,CRISPR/Cas9 gene editing technology is currently the most widely used technology.It saves time and effort in targeting exogenous genes,and greatly reduces the cost of gene editing.However,CRISPR/Cas9 technology still achieves the integration of multiple copies of exogenous genes.There are technical difficulties.The Phi C31 integrase itself can mediate specific att sites to achieve irreversible insertion of foreign genes,and is not limited to single copy integration.Combining gene editing technology represented by CRISPR/Cas9 technology with Phi C31 integrase provides a new idea for the integration of multiple copies of foreign genes.This study intends to use CRISPR/Cas9 gene editing technology to integrate 3×att P,6×att P and 9×att P sequences into the goat ROSA26 locus,and obtain corresponding positive clones through drug screening and Junction PCR identification and study their functions.The main results are as follows: sg RNA is designed for the ROSA26 locus,and a target site with high cleavage efficiency in vitro is obtained.The donor vectors ROSA26-PURO-3×att P,ROSA26-PURO-6×att P and ROSA26-PURO-9×att P with att P sequence were successfully constructed.Different donor vectors and Cas9 eukaryotic targeting vectors were co-transfected into dairy goat fibroblasts.Att P stable integrated positive clones were screened with puromycin,and Junction PCR was used to identify accurately integrated positive monoclonal cells.The att B-Neo-m Cherry-EF1α donor vector and p CMVInt-Phic31 expression vector were constructed to conduct a second electrotransformation on the positive cloned cells integrated with att P series.Edu detected att P stable integration positive monoclonal and F3 dairy goat fibroblasts.The comparison showed that the cloned cells with higher proliferation activity can be used for subsequent somatic cell nuclear transplantation in dairy goats for the next step of verification.Although this experiment did not obtain an effective foreign gene integration efficiency,the combination of CRISPR/Cas9 technology and Phi C31 integrase provides a new research direction for exploring the integration of foreign genes in multiple copies,and is also useful for mammary gland bioreactors.Development and the exploration of livestock genomes provide new ideas.