원문정보
초록
영어
Genome editing is one of the most demanding tools in mammalian cells to unravel the role of genes and to implement gene therapy of genetic diseases. Zinc finger nuclease is an engineered restriction enzyme which is constructed by combining an array of zinc finger DNA-binding motifs in the N-terminus and the FokΙ endonuclease in C-terminus. Almost every ZFN recognizes three consecutive nucleotides starting with guanine in a context-dependent manner, thereby limiting the use of ZFN in genome editing. To address this challenge, we replaced ZFNs with a gene-specific nucleotides, which were connected to FokΙ nuclease by using (his)6-tag and GS linkers. We tested in vitro whether this engineered FokΙ shows a site-specific nuclease activity. For this, the engineered FokI and nucleotide complex was incubated with template DNA which was produced from 70-mer and 100-mer nucleotides base-paired each other. The results indicate that the complex showed a site-specific nuclease activity, which suggests that the nucleotide-based FokΙ can be used for the universal genome editing.