Highly Efficient Gene Editting Tools-mediated Transgene Knockin At The H11Locus In Pigs

Transgenic pig plays an important role in agriculture and biomedicine. On theone hand, for pig is an important livestock, transgenic techology bring a great helpon meat quality improvement, pork production performance, environmentalprotection breeding and disease resistant breeding. On the other hand, pig is similarto human. For they have the similar size of organs, xenotransplantation brcomepossible. And pig and human also have the similar structure of tissues, they are usedin xenotransplantation, diabetes, atherosclerosis and Alzheimer’s disease research.Although transgenic pigs have been widely used, it is still limited.The efficiency and the integration site of the transgene uncertainty are the mainreasons. To solve this problem, Liangxue Lai’ group identify the pig Rosa26sitesuccessful. They insert loxP into this locus and finished the gene substitutionsuccessful. The foreign gene like RFP can express well in this locus. But Rosa26isgene that express abroad, and it has its own Rosa26promotor. Tissue-specificexpression is often needed, and Rosa26locus is hard to hit this target.In2010,Simon Hippenmeyer and his team in Stanford University identify agood locus in chromosome11of mice, which named hipp11(H11). H11locuslocated between the exon19of Eif4enif1and the exon9of Drg1gene. This newgenomic “safe harbor” locus, named pH11, which enables stable and robusttransgene expression and no gene silencing happend.According to the human and mice H11locus, we identify the pig H11locus in chromosome14. We did not find any SNP in H11locus in5varieties of pig. In oderto inseart the efficiently, we designed and construct five pairs of TALENs, one pairof CRISPR/Cas9n and two CRISPR/Cas9. And then their efficient weretested testedby T7EI digestion method. We found CRISPR/Cas9was the most efficient.According to the sequence test method, the result showed that the efficient ofCas9-H11-g1reached64%.23predicted off-target sites were found according to thetwo CRISPR/Cas9in pig genome. And all these sites were amplified from the DNAisolate from Cells transfected with CRISPR/Cas9, and then they were digested byT7EI. No off-target evidence were found here.Two targeting vectors were designed according to screening methods. Wefinished our cell screening works by postive/negtive screening method, postivescreening method and no drug screening methodseparately. We got positive clonessuccessful from all three screening methods. The efficient of postive/negtivescreening method reached23%, the efficient of postive screening method reached54%, the efficient of no drug screening method reached6%. The no drug screeningmethod is most efficient in cell screening.In order to verify theforeign gene inserted into pH11locus can express well, weuse the GFP as report gene which promote by CMV promotor. Our results showedthat GFP express well in all the positive cell clones and embryo. And then one clonepig was created from positive cell clone by somatic cell cloning technology. Ourresults show that GFP can be expressed well in heart, liver, spleen, lung, kidney andmuscle tissue. So we consider pH11allow foreign gene express well.Our work identified the pig H11locus firstly. Furthermore, we construct kindsof gene editting tools, and found CRISPR/Cas9systerm were the most efficient foreditting. And then, we finished our cell screening by three kind of screeningmethods using CRISPR/Cas9. Our results showed that positive screening methodwas the most efficient. We were the first time got the knock-in pig cells by no drugscreening method. We successfully inserted a gene fragment larger than9kb at the pH11locus using the CRISPR/Cas9system. And at last our results confirmed thatthe foreign gene inserted into pH11locus can express well in all cell, embryo andindividual levels.In summary, this study created an efficient knock-in systerm in pH11locus viagene editting method. This systerm will offer a safe and efficient platform fortransgenic pig produce.