Optimization Of CRISPR/Cas9 System To Improve Genome Editing Efficiency Of Disease-Resistant Cloned Cattle

Mastitis and bovine tuberculosis,as major diseases that harm the dairy industry,can cause a series of problems such as milk yield decrease,milk quality decrease and treatment cost increase.The pathogens that cause mastitis and bovine tuberculosis can be transmitted through contaminated,unpasteurized milk,posing a serious threat to human health.At present,there is a lack of effective means to control these two diseases in dairy farming in China.There are studies that show that overexpression of natural resistance-associated macrophage protein 1(NRAMP1)gene and insertion of mouse intracellular pathogen resistance 1(Ipr1)gene improved the resistance of transgenic cattle to tuberculosis.Geneedited cattle with site-directed integration of the human Lysozyme(h LYZ)gene significantly resist infection with pathogens that cause mastitis.Therefore,the production of diseaseresistant gene-edited cattle provides a useful idea for reducing the harm caused by mastitis and bovine tuberculosis.However,the efficiency of homology-directed repair(HDR)mediated site-specific integration of foreign genes in these studies was low,which limited the development of new breeds of gene-edited cattle.Therefore,improving the efficiency of exogenous gene insertion can greatly promote the application of gene editing technology in cattle disease resistance breeding.This study aims to optimize the CRISPR/Cas9 system to improve the efficiency of site-specific integration of disease resistance genes into holstein dairy cow genome,and provide technical support for the production of disease resistance gene editing cattle and subsequent propagation.The main research is as follows:1.A reporting system was constructed to detect the site-specific integration of foreign genes mediated by homology-mediated end-joining(HMEJ)-,microhomology-mediated end-joining(MMEJ)-,non-homologous end-joining(NHEJ)-and HDR-based strategies at bovine MS locus(the region between methionine adenosyltransferase 1a and surfactant protein-A1 genes on chromosome 28)and identified bovine ROSA26 locus,respectively.The results showed that the efficiency of the HMEJ-based strategy was significantly higher than that of the NHEJ-,MMEJ-and HDR-based strategies in the two locus.The genome editing system for bovine fetal fibroblasts was optimized by using the method of ROSA26 gene endogenous promoter and the HMEJ-based strategy to efficiently screen positive cells for site-specific integration of foreign genes.2.A traffic light reporter system was constructed using lentiviral vectors to simultaneously detect HDR and non-homologous end-joining(NHEJ)frequencies.The effect of small molecule drugs RS-1,SCR7 and NU7441 on HDR mediated exogenous gene integration was evaluated by the reporting system and the way of screening positive clones.3.Three NRAMP1 gene-edited bovine fetuses and h LYZ gene-edited bovine fetuses were successfully produced by using the above optimized gene-edited system combined with nuclear transfer technology.The method of exogenous promoter to drive marker gene expression combined with HMEJ strategy also effectively screened positive cells with Ipr1 gene site-specific integration,and finally obtained a gene-edited calf with site-specific insertion and normal expression of disease-resistant gene.In summary,this study found that the efficiency of exogenous gene insertion dependent on HMEJ was significantly higher than that of the NHEJ-,MMEJ-and HDR-based strategies;Using HMEJ mediated site-specific integration of resistance genes,we obtained the gene-edited cattle at b ROSA26 locus,which provided valuable materials for disease resistance breeding.