Construction Of Zinc Finger Nucleases And Its Application Of Animal Genomic Editing

Precisely and highly efficient modification of endogenous genes is one of the mostimportant issues in the development of transgenic technology. Two major traditional methods,spontaneous recombination with extremely low efficiency (1×106) and retrovirus mediatedgene target with concern of safety are limited in wide application and development oftransgenic breeding technology. As an artificial nuclease, zinc finger nuclease (ZFN) iscomposed of a DNA binding domain of zinc finger protein (ZFP) and non-specific DNAcleavage domain of FokⅠ. A pair of ZFN as a dimer cut target locus in genome, resulting inDSBs. In the process of DSBs repair, genomic editing is accomplished, for instance, targetedgene knock-in, gene knock-out, gene correction, gene disruption.The cleavage specificity efficiency of ZFN is largely determined by zinc finger proteins.Currently, zinc finger proteins were obtained via modular assembly or library screeningsystem, then fused to FokⅠ to generate active ZFNs target sites of interest. Whereas, ZFNsalways failed to cut target site, even the ZFPs had ability to recognize and bind DNA targetsites. Thus, obtaining active ZFNs is an urgent issue about this technology. In addition, how toenrich positive cells with gene-modification and obtain transgenic cell lines are crucial to theapplication of ZFNs.Previous studies all focused on achievement of specific ZFPs. In this study, we created ayeast-based screening system to obtain ZFNs directly. Goat alpha S1-casein gene as a safeharbor and target gene for generation of transgenic cell lines. According to the target sequence,active ZFNs were screened from randomized ZFNs libraries via yeast-based screening system.Then, activities of screened ZFNs were validated in human HEK293T cells. Finally, ZFNs cutalpha S1-casein gene in target cells. Under the pressure of puromycin, positive cells wereenriched after ZFN treatment and cells with target gene modified were achieved.1. According to the OPEN method, three randomized ZFN libraries were constructedusing PCR with randomized primers. In each library, sequences encoding the key amino acidsin one motif were randomized; the other two zinc finger motifs were maintained with thestandard frame. On the level of plasmid, the complexity of each library was about106~107independent colonies. Meanwhile, nine reporter vectors were constructed harboring potential target sequence of goat α S1-casein gene.2. The yeast screening system was based on Gal4protein, in which three-step screeninginvolved. The first step screening aimed to enrich six single finger motif bind to each triplet(3-bp). Enriched single motifs were assembled together to generate two reconstructed ZFNlibraries. ZFNs target9-bp left or right half sites were obtained through the second stepscreening. Finally, three pairs of active ZFNs target full site were achieved via the third stepscreening.3. To detect activities of screened ZFN pairs, ZFN mammalian expression vectorspST-ZFNL, pST-ZFNR and reporter plasmid containing pRFP-EGFP-BS two fluorescentgenes were constructed. ZFNs expression vectors and reporter plasmids were co-transfectedinto HEK293T cells. Upon ZFNs cut target site in EGFP gene, which was restored duringDSBs repair and green fluorescence cells were observed. The rate of EGFP-positive cellsindicated cleavage activity of ZFNs. Then, the three pairs of ZFNs activities were8.9%,9.3%,5.0%, respectively.4. Active ZFNs worked in target cells—goat mammalian epithelial cells. ZFNsexpression vectors and another reporter vector pB-CBA-Puro-BS were co-electroporated intotarget cells. Upon ZFN-mediated cleavage at target site on reporter, puromycin resistant genewas restored to enrich positive cells. Thus, clones survived in the medium with highconcentration of puromycin. Genomes of treated cell were isolated, and target sequences wereamplified using PCR. T7endonuclease I assay was performed to analyze mutation rate oftarget gene. No insertions or deletions were detected without puromycin enrichment system.Nevertheless, mutations were observed under puromycin selection pressure in the enrichmenteffect of reporter system. Finally, gene mutation rates of three pairs ZFNs generated in targetcells were9.4%,15.9%,4.1%, respectively.5. ZFN consists of two separably functional domains, DNA banding domain (zinc fingerprotein) and nuclease domain (FokⅠ). The bait protein is fused with a zinc finger protein anda prey is expressed as fusion protein with FokⅠ. A unique intermediate reporter vector isdesigned such that Gal4transcription factor gene is disrupted by the ZFN target sequenceflanked by two short repeats. The interaction of bait and prey proteins reconstitutes the ZFNfunction, resulting in double strand break (DSB) on its target sequence. The DSB activatescellular DNA repair system, leading to restoring a functional Gal4transcriptional factor whichcan activate reporter genes, His3, Ade2, LacZ in AH109yeast strain. This system can betheoretically used to any type of cellular proteins. Additionally, by using intermediate reportergene Gal4, the interaction signal is amplified. This novel system will provide an additionaltool for the technologies of protein interactomics.