Construction Of Artificial Zinc Finger Libraries And Application In Screening Zinc Finger Nucleases

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain (Fok I). ZFN can be used to precisely alter the genomes of higher organisms. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. Therefore, the key step in construction of zinc finger nuclease is the selection of specific zinc finger proteins.The oligomerized pool engineering (OPEN) approach were applyed to select specific zinc finger proteins in this reseach. Key amino acids at positions ?1, +1, +2, +3, +4, +5 and +6 relative to the start of theα-helix of each zinc finger motif contribute to most of the sequence-specific interactions with the DNA site. Based on three-finger ZFP from a framework Zif2687, the sequences coding seven key amino acids were randomized by Cassette Mutagenesis. Three zinc finger random libraries were constructed subsequently. The ZFPs were fused to the non- specific endonuclease domain of Fok I to form ZFNs. Then the activity of ZFNs was examined in yeast validation system. The results as follows:1. Three zinc finger random libraries (Randomized zinc finger 1 library, Randomized zinc finger 2 library and Randomized zinc finger 3 library) were constructed. Human DYRK1A gene was exemplified to establish a technology platform for screening of ZFPs. The zinc-finger targeter (ZiFiT) program was used to identify potential full ZFN sites of genomic sequence of human DYRK1A gene. The target sequence is 5'-TCGACCACCAGCATC- GGCACAGTGGTGGGCAG-3'. A serial reporter plasmids were constructed subsequently. Three zinc finger random libraries were used to screen specific zinc finger proteins binding DYRK1A gene. Fifty zinc finger proteins were isolated by bacterial two hybrid. 2. We demonstrated a rapid and precise system to test ZFNs activity in yeast cells. The express plamids pTrp-ADH1-ZFNLå'ŒpLeu-ADH1-ZFNR were constructed by by fusing ZFP to a Fok I. In view of repair mechanisms of Double-stranded DNA break in yeast cells, The repoter plasmid pUra-KanMX4-ZFBS were constructed. 90% of the target site in reporter plasmid were cleaved by ZFN, and repaired by mechanism of homologous recombination in yeast JMY31. The results supported the high efficiency of OPEN appoach. Preliminary tests of ZFN activity using the system will simplify experimental operations, improve the success rate of experiments.