人工锌指蛋白随机库的构建及其在锌指核酸酶筛选中的应用

利用开源式(Oligomerized pool engineering,OPEN)方法筛选具有较高特异性和亲和性的锌指蛋白(Zinc finger protein,ZFP).以已知三锌指蛋白为框架,使单个锌指关键位点氨基酸编码序列产生随机突变,构建3个人工锌指蛋白随机库,建立和完善应用人工锌指蛋白随机库筛选特异性锌指蛋白的技术平台.以人DYRK1A基因为例,测试和验证该技术平台的可行性和效率,分别获得对DYRK1A基因靶序列中左侧和右侧位点具有较高亲和性的50个三锌指蛋白；然后将得到的锌指蛋白与非限制性核酸内切酶FokⅠ连接,构建DYRK1A锌指核酸酶(Zinc finger nucleases,ZFN),应用酵母验证系统测试DYRK1A锌指核酸酶的活性,结果表明,90％的组装锌指核酸酶能够在靶位点进行切割.

Construction of Artificial Zinc Finger Libraries and Application in Screening Zinc Finger Nucleases

The oligomerized pool engineering(OPEN) approach was applied to select specific zinc finger proteins(ZFP) with high affinity.We constructed three zinc finger random libraries,each based on a synthetic "framework" ZFP.In each library,recognition helix residues-1,1,2,3,4,5,and 6 from a single finger were randomized by cassette mutagenesis.The three zinc finger random libraries were used to screen specific zinc finger proteins.Human DYRK1A gene was exemplified to examine the feasibility and efficiency of the technology platform for screening of zinc finger proteins.As a result 5o zinc finger proteins with higher affinity to the recognition sites of DYRK1A gene were obtained.The zinc finger proteins were fused to the non-specific endonuclease domain of Fok I to form zinc finger nucleases.Then the activity of zinc finger nucleases was examined in yeast validation system.The result showd that 90% zinc finger nucleases worked well in yeast cells.This research provided the technique support for the application of zinc finger nucleases to the editing of the genome.