Site-specific Editing Of Pig Genome Using CRISPR/Cas9 And Dysregulation Of DNA Methylation In Cloned Piglets

Pig is an important livestock that not only provides diet protein but also is an appropriate medicine model, besides it could be donor for organ transplantation to humans. Sow milk is the main food source for piglets. Lactoferrine provides anti-microbial, antiviral and absorbing iron to the piglets. While its concentration is too low that cannot satisfy the need of piglets. Improving the content of lactoferrine will benefit the health of piglets. Traditional breeding is difficult to improve the quality of sow milk. Transgenic technology provides the fast method to improve our interesting traits. While conventional transgenic technology produces animals via random insertion, and it cannot ensure the gene expression and its safety. In recent years, CRISPR/Cas9 appears to edit exogenous genes, and now it is used by many scientists. CRISPR/Cas9 can lead to the double strand break of the target site, and then the efficiency of homologous recombination repair will increase thousands times comparing to traditional methods. So the CRISPR/Cas9 will help us produce the transgenic pigs via homologous recombination. However, the efficiency of the CRISPR/Cas9 is still low and has off-target effects. So we also conducted the experiments to improve its efficiency and reduce the off-target. Somatic Cell Nuclear Transfer(SCNT) is an important step to produce the transgenic animals, while the mechanism of reprogramming in SCNT is still unknown. The efficiency of SCNT is still low(less than 5%) and many of the cloned animals have physical defect. So in our study, we also analyze the DNA methylation and gene expression in the whole genome for the abnormal cloned pigs to provide some clues for explaining the abnormal phenotype in cloned pigs. The results are as follows:(1) We designed effective sg RNAs targeting the CSN1S1 gene. The efficiency of CRISPR/Cas9 varies in different transfection methods. The efficiency of Nucleofaction is 3-4 times than lipidosome. Without the homology template, the double strand break of DNA is repaired by non-homologous end joining which produce indel in the target site.(2) In order to make a transgenic pig with high level of lactoferrin in milk specifically, we tried to integrate the LTF CDS into the stop codon of CSN1S1 gene blanked by a short peptide adaptor of 2A. Self-cleaving of 2A was verified using Western blot, and the efficiency reached 100%. After selection with appropriate primers, we got the positive cells which the LTF was inserted into the stop codon of CSN1S1 gene blanked by a short peptide adaptor of 2A. The cells would be used as the donor cells for SCNT.(3) To improve the efficiency of gene targeting using the CRISPR/Cas9, we designed a vector containing the Cas9 gene and the EGFP gene. After selection with FACS, the targeting efficiency could reach 100%, which provided a useful tool to study the gene function. Five or eight sgRNAs targeting the same gene were transfected into the cells, and the targeting efficiency was tested via western blot. It was interesting to find the level of protein expression is lower than single sg RNA, while the mRNA was in the same level.(4) The truncated sgRNA may reduce the off-target effect induced by CRISPR/Cas9 reported by other group. To test this hypothesis, we selected eight genes in human genome, and calculated the off-target in truncated sgRNA. The potential off-target site increased in the truncated sgRNA, but in some off-target site, the efficiency of truncated sgRNA reduced. While in other off-target site, the efficiency is still higher than 20 bp sgRNA.(5) From the analysis of DNA methylation in the cloned pigs, the hypomethylated regions were more than hypermethylated regions in the abnormal cloned pigs. In the CpG islands, the level of DNA methylation of the abnormal cloned pigs was higher than that in the normal cloned pigs. Some differentially methylated genes owned both the hypomethylated regions and the hypermethylated regions. We got more than 1700 differentially expressed genes between the two groups,and more than 1500 genes were up regulated. 243 genes were altered in both the DNA methylation and the gene expression.MAPK signaling pathway was one of the different pathways reported by other group.(6) We combined the data of DNA methylation and the gene expression, and found the DNA methylation and gene expression around the TSS was negative correlation. In normal cloned piglets, the DNA methylation and gene expression in gene body was positive correlation. The relationship of the DNA methylation and gene expreesion in abnormal piglets was not clear as the normal cloned, which might affect the normal development.