Optimizing CRISPR/Cas9 System To Improve Gene Targeting Efficiency Of ROSA26 Locus In Dairy Goat

Animal mammary gland bioreactor is a technology that uses transgenic technology to introduce foreign genes into animal genome and obtain transgenic animals with mammary gland-specific expression of target proteins.At present,a variety of medical protein products have been approved and put on the market.It has created great economic value.At the same time,dairy goats are widely used in mammary gland bioreactors because of their suitable size,short pregnancy and rich milk secretion.However,the technology of preparing transgenic animals is still immature,and the transgenic animals obtained by somatic cell nuclear transfer have low pregnancy rate,high mortality and low expression caused by position effect.These factors seriously affect the application and development of animal mammary gland bioreactor.Based on the above,the selection of efficient gene targeting technology and safe targeting sites are two important factors to ensure the efficient expression of the target protein in the mammary gland and to improve the preparation efficiency of transgenic animals.CRISPR/Cas9 technology is the third-generation gene editing technology after ZFNs and TALENs.With high efficiency,low cost and easy operation,CRISPR/Cas9 technology can be widely used in many species.The ROSA26 locus is considered to be the closest to the definition of safe haven for gene insertion,which is considered to be a friendly site for the integration of foreign genes in many species.In this study,CRISPR/Cas9 technology was used for the first time to target the gROSA26 loci of primary dairy goat cells with different strategies,in order to obtain a more efficient and safe gene targeting system for the preparation of dairy goat mammary gland bioreactor.The experiment was to use dairy goat mammary epithelial cells as experimental materials,and the gROSA26 transcript sequence was obtained by RACE technique.The possibility of screening marker gene to obtain monoclonal cells was determined by identifying the core promoter region and its promoter intensity.Then the sgRNA design website CRISPOR was used to screen the target sites with the least potential miss sites in the first intron region of gROSA26.SSA double fluorescence was used to detect the cleavage efficiency of different sites in vitro to obtain sgRNA with higher shooting efficiency and lower miss effect.Furthermore,four donor vector reporter plasmids of HDR,HMEJ,NHEJ and MMEJ were constructed by using different DSB repair mechanisms.These plasmids and Cas9 targeting vectors were cotransfected into dairy goat mammary epithelial cells to screen the positive clones using endogenous promoters to express screening marker genes.It is confirmed that the target shooting efficiency under the HMEJ strategy is the highest.Finally,the HMEJ donor vector was constructed,in which the screening marker was initiated by gROSA26 endogenous,and the human lysozyme was initiated specifically by mammary gland.The positive clones with site-specific integration of human lysozyme gene at gROSA26 site were screened.As a result,a CRISPR/Cas9 gene targeting system with higher integration efficiency and more stable expression of foreign genes was obtained,which laid a foundation for the follow-up large-scale application of dairy goat mammary gland bioreactor to produce pharmaceutical proteins.