Optimizing The Screening System Of Zinc Finger Nucleases Targeted Pig CFTR、MSTN、ApoE Gene

As one of the genome modification tools, zinc finger nucleases(ZFNs) have been widelyused in many fields, the most prominent and potential field is gene therapy. There have a lotof advantages that ZFNs compare with TALENs and CRISPR/Cas9in the gene therapy field,such as the lower efficiency of off-target and less-cell toxicity. Now ZFNs has already beenused in clinical trials in the field of gene therapy. However, there are some disadvantagessuch as the complicated zinc finger protein assembly systems and time-consuming. Thus,how to simplify the work and save the time are very meaningful.ZFNs are composed of a designed polymeric zinc finger proteins(ZFPs) and anon-specific DNA cleavage domain of FokⅠ restriction endonuclease, the specificity ofZFNs is largely determined by ZFPs，which could recognize and bind to specific DNA targetsequence. Two Fok Ⅰ dimerized can induce secquence-specific DNA double-strandbreaks(DBSs), which could be repaired by the homologous recombination (HR) with donorDNA or the error-prone non-homologous end joining (NHEJ) system to yield insertion and/ordeletion(indel) mutations at targeted genomic loci in vivo. The most important of ZFNtechnique is obtaining ZFP with high affinity. Current studies about the methods ofconstructing ZFNs are module assembly (MA), context-dependent assembly(CoDA) andOligomerized Pool Engineering(OPEN). MA is a quick and straightforward method, but itreported low success rate and ignored the context-depended effect between fingers. CoDA isa quick and easy method, but relatively important finger2is sonstant, the limited informationof known zinc finger arrays leads to failure of finding efficient ZFNs to some target genes.Although the OPEN method has no shortcomings as what mentioned before, but thecomplicated screening process when construction of ZFNs limited its easy-use in manylaboratoraies. Therefore, the key is how to solve these problems to improve ZFN technology.Comparing the advantages and disadvantages of the methods of construction ZFNs, weimprove the screening system in OPEN method. Open employs a two-step screening, the firststep is to establish pre-screened pools of single zinc fingers targeting a specific triplet from apool of nearly randomized oligos by using a phage-display system, the second step is toassemble3pre-screened pools into a ZFP pool to perfrom the second screening, they are allneed lots of time and labor. Analyzed the method of OPEN screening in the relevantliteratures-bacterial two hybrid system, yeast two-hybrid system are used to screen for the active ZFNs. The bacterial system offers a number of potentially significant advantages overexising yeast-based two-hybrid method due to the higher transformation efficiency and fastergrowth rate observed with Escherichia coli. The bacterial two hybrid system in traditionalOPEN method is based on a blue/white screening which the LacZ as a reporter gene,researchers need to pick many single colonies to identify. Previous studies demonstrated that3-AT which is a competitive inhibitor of His3, can be used to titrate the level of His3expression required for growth on medium lacing histidine(His). Therefore we inserted theHis3reporter gene in the downstream of the DNA target sites, the colonies harboring activeZFP can live in the synthetic dropout minimal base with3-AT. Because when the ZFP bindingthe target DNA, the transcription factor fused by the ZFP can turn on the His3geneexpression. This method can inhibit the basal expression of His in the presence of3-AT, andimprove the efficiency of obtaining the positive colonies. Then we constructed expressionvector to test the activity of the ZFNs based on yeast, and simultaneously we isolateed theactive ZFNs and tested the efficiency.Finally, we obtained nine pairs effective ZFNs of three genes, and cost much less timethan before. Screening the ZFNs from the pools just cost5weeks. Compared with theblue/white screening we saved time a lot. This study indicated that the improve screeningsystem cost much less time to achieve active ZFNs on purpose. When we need constructZFNs, the optimized OPEN method makes the process of obtaining ZFNs much easier andmore convenient.