Knockout Of Mice Vitamin D Receptor Gene Via CRISPR/Cas9 System

The ability of gene knock-out or gene knock-in is the most effective tool for gene function research, but achieving high specific targeted genome editingproducts has always been a bottleneck problem that limited the rapid development of biological research. The traditional gene targeting technology was based on homologous recombination(HR), unfortunately, the efficiency of HR is extremely low, and the cost is very high, so it is limited in wide application. Since the discovery that the efficiency of homologous recombination(HR) could be boosted in cell by introducing site specific DNA double-strand breaks(DSBs), the research and development of artificial nucleases that can cleavage DNA at specific sites was a key point in biotechnology. CRISPR/Cas9 system is the third generation genome editing tool after ZFNs and TALENs, and it was an RNA-guided DNA endonuclease that can introduce DSBs, and DSBs were efficiently repaired by HR using a donor DNA as template or by error-prone non-homologous end-joining(NHEJ), leading to targeted genetic modifications in cells. CRISPR/Cas9 can be customized by replacing guide RNAs to achieve different genes modification, making it much more affordable and scalable.Vitamin D receptor(VDR) is a nuclear transcription factor responsible for the biological activity of vitamin D by binding 1,25-dihydroxyvitamin D(1,25D). Present in a diverse range of tissues, VDR has the ability to exert an extensive biological response via regulation of gene transcription and stimulation of intra-cellular signaling pathways. VDR has been playing many roles in calcium homeostasis, inhibition of cellular proliferation, stimulation of cell maturation and health, which may involve various tissues including skin, the immune cells, colonic, breast and prostate cells. In this study, VDRT1 and VDRT2 were selected in conserved coding region of VDR gene according to the design principle of CRISPR/Cas9, and constructed two sets of CRISPR/Cas9 targeting plasmids and report vectors. The activity of CRISPR/Cas9 systems was detected in HEK293 T and C2C12 cells. At last, we generated VDR knockout mice via zygote injection of CRISPR/Cas9 System, and the partial function of VDR was verified in VDR knockout mice. Moreover, we explored the technology program of Cas9 transgenic mice generationwhich was mediated by lentivirus injection in testis.(1) To achieve targeted modifications at the same site in both human and mice genomes, we designed two target sites in conserved coding regions of vitamin D receptor(VDR) gene by online software. We respectively constructed two CRISPRR/Cas9 targeting plasmids and corresponding report vectors via direct annealing of two oligonucleotides. The purpose of report vectors was to detecte the activity of CRISPR/Cas9 and to enrich positive cellcolonies.(2) In order to test the cleavage efficiency of CRISPR/Cas9 and the ability of large fragment deletion on target sites in VDR, the CRISPR/Cas9 systems of VDRT1 and VDRT2 were separately transfected or co-transfected into human 293 T cells and mice C2C12 cells. The results shown that the mutation frequencies of these two VDR sites in HEK293 T cells were 36% and 31%, and the cleavage efficiency of the VT1 and VT2 sites inenriched C2C12 cells were 26% and 22%. The research showed that large fragment could be deleted in VDR gene using CRISPR/Cas9, and the frequencies of large fragment deletions can reach 10%.(3) To generate VDR knock-out mice, the Cas9 mRNA and sgRNA mixture targeting VDRT1 and VDRT2 was injected into one-cell-stage embryos of C57BL/6J mice. Through sequencing analysis, we had confirmed that 12 VDR knockout mice were produced, and 8VDR knockout mice were biallelic gene knockout,and 5 knockout mice had same indels. To test whether off-target mutations occurred in VDR knockout mice, 6 potential off-target sites were selected for detection,the results indicated that two sites OT1 and OT5 with off-target modifications in mice genome.(4) In order to further clarify the effect of VDR targeting, we analyzed the expression of VDR and VDR related genes in different tissue via immunofluorescence histochemistry and qPCR. The result of qPCR showed that VDR was present in different tissues, and the transcriptional level of VDR was remarkably increased in some tissues of VDR knockout mice. However, the results of immunofluorescence histochemistry showed that there was no VDR expression in different tissues of VDR knockout mice. And then, we speculated that possibly there was a mechanism of negative feedback in VDR expression. And the results of VDR related gene indicated that VDR regulates many genes expression at different levels in various tissues. The phenotypic properties of knockout mice had shown that VDR mutation could affect the grown and development of bone and hair, and maybe also affect the reproductive ability.(5) We injected eGFP or Cas9 lentivirus into the inter-tubular space of mice testis by two injection methods, to infect the micegermcells, and obtained the genetic modified sperms, to generate transgenic mice. The results had shown that the efficiency of transgenic mice production was much different between the following two methods. The efficiency of accurate injection of seminiferoustubules reached above 50%, and the efficiency of random multi-point injection method was only 12.5%...