Research On The Preparation Of Imprinted Gene Knockout Mice Via CRISPR/Cas9

Genome editing techniques, especially the emerging CRISPR/Cas9 system, can modify the genome efficiently in many species, and have been shown great research value in the field of life sciences and medicine. Traditional gene targeting technique relies on homologous recombination of DNA fragments and has low efficiency(less than 10-6). Comparing with the complex procedures of ZFNs and TALENs,CRISPR/Cas9 system, by which target genes can be edited just using guide RNA and Cas9 protein, has advantage of simple assembly, high efficiency, low cost and so on. The purpose of the experiment is to knockout target gene at the cellular and embryonic level via CRISPR/Cas9 system and obtain the gene knockout mice.In this study the maternal imprinted gene Kcnq1ot1 was selected and designed to be knocked-out,which is located in the imprinted gene cluster of Kcnq1ot1 and plays a important role in regulating the expression of imprinted genes. Our first technical route used the method of ES cell-mediated blastocyst injection to prepare the transgenic mice. The strategy was to knockout the promoter and transcription initiation site of Kcnq1ot1 gene,and knockin puro-eGFP. We first searched three targeting sites in 5' and 3'ends of the knockout fragments and constructed six gRNA vectors and a targeting vector(Kcnq1ot1-puroeGFP-p Galk). Then four vectors including the targeting vector, Cas9 expression vector, 5'gRNA-1 and3'gRNA-1 were co-transfected into mouse ES cells. Finally, the ES cell lines with kcnq1ot1 gene monallelic knocked-out were successfully obtained. We conducted blastocyst injection using the CD1 mice as the embryos provider, totally injected 1836 blastocysts, transplanted 826 embryos and obtained 138 mice(the birth rate is 16.7%). At last, we successfully obtained four chimeric mice, however, none is germline chimera.The pronuclear microinjection was used to prepare the transgenic mice in our second strategy. The kcnq1ot1 gene was knocked-out like the first strategy but the mCherry gene was knocked-in. In order to improve the efficiency of homologous recombination, we increased the length of homologous arms to1.5kb and constructed a new targeting vector(Kcnq1ot1-mCherry-pGEM-3CZF). We detected the efficiency of homologous recombination of the 6 gRNA sites at the level of plasmid through flowcytometry. Then the 5'gRNA-2, 3'gRNA-3 with high efficiency of HR, targeting vector and Cas9 expression vector were injected into the male pronucleus of fertilized eggs. Using D6B2F1 mice as the embryos provider, we totally injected 583 embryos, transplanted 327 embryos and obtained 66 mice(the birth rate is 20%). We failed to obtain the positive mice due to the low efficiency of homologous recombination. Next, we planned to increase the number of injected embryos in order to obtain the gene knockout mice.