猪CFTR基因特异性锌指核酸酶的筛选与鉴定

Screening and validation of specific zinc finger nucleases targeted pig CFTR gene

【目的】获得靶向猪囊性纤维化跨膜传导调节因子(Cystic fibrosis transmembrane conductance regu-lator,CFTR)基因的特异锌指核酸酶(Zinc finger nuclease,ZFN),为建立CFTR基因敲除猪细胞系提供技术支持。【方法】通过开源式(Oligomerized pool engineering,OPEN)方法筛选,首先以已知三锌指蛋白为框架,随机突变单个锌指关键位点氨基酸编码序列,建立人工三锌指蛋白随机库;然后应用细菌双杂交技术,从库中筛选出能够结合CFTR基因靶位点的三锌指蛋白;最后将获得的锌指蛋白与非限制性核酸内切酶FokⅠ组装成特异ZFN,通过酵母验证体系,检测ZFN靶向切割其识别序列的效率。【结果】获得3个人工三锌指蛋白随机库,每个库约含有2×106个单克隆,通过2轮细菌双杂交筛选,分别获得48个针对CFTR基因左右两侧靶位点的三锌指蛋白。ZFN活性验证结果表明,约90%的ZFN能够在酵母细胞内实现靶向切割,获得了高效特异的ZFN。【结论】获得了猪CFTR基因高效特异的ZFN。

【Objective】Specific zinc finger nucleases（ZFN）were screened to target pig cystic fibrosis transmembrane conductance regulator（CFTR）gene for the preparation of generating CFTR gene knockout pig cell line.【Method】Upon the oligomerized pool engineering（OPEN）strategy,firstly,three libraries of 3-zinc finger proteins were constructed.The DNA sequences of recognition amino acid residues of a finger motif were randomized by cassette mutagenesis.Secondly,bacterial twohybrid（B2H）system was used to screen for ZFPs from the three zinc finger protein libraries.Finally,selected ZFPs were fused to FokⅠdomain to generate specific ZFN for subsequent validation of ZFN cleavage activity in yeast.【Result】Three artificial randomly libraries of 3-zinc finger proteins were obtained,and each contained about 2×106 colonies.The three zinc finger random libraries were used to screen specific zinc finger proteins by two steps of B2H.Forty eight specific ZFPs with high affinity to the left and right half sites of CFTR target site were gained,respectively.The result of ZFN activity validation in yeast demonstrated that 90% of screened ZFN had the ability to cleave CFTR target site,and specific ZFN were achieved via this strategy.【Conclusion】The ZFN with high affinity and efficiency will be applied to target pig CFTR gene resulting in generation of CFTR gene knockout pig cell lines,which is fundamental to make the disease model for gene therapy and drug discovery.