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The effect of the length of donor homologous arm on the efficiency of ZFN-induced homologous recombination
Pages: 100-107
Year: Issue:  4
Journal: Acta Scientiarum Naturalium Universitatis Sunyatseni

Keyword:  zinc finger nucleasedouble-strand breakhomologous recombinationdonorhomologous arm;
Abstract: Zinc finger nuclease (ZFN)is composed of an engineered site-specific Cys2-His2 zinc finger domain and the nonspecific restriction enzyme Fok I cleavage domain,which is able to cut at a specified genomic locus to generate double-strand break (DSB)of DNA.The DSBs induced by ZFN are subse-quently repaired through two different DNA repair mechanisms,either non-homologous end-joining (NHEJ)or homology-directed recombination (HDR).NHEJ is prone to introduce sequence insertions or deletions (indels),and can therefore produce frameshifts in open reading frames and gene loss of func-tion.HDR requires a donor template with the sequence similar to the genome to mend a lesion.By intro-ducing a DNA donor with desired modifications,precise genomic modifications can be achieved at a fre-quency improved 102 -104-fold as compared to the traditional gene targeting method.Currently,most studies have focused on screening ZFN with higher activity,and improving the delivery efficiency of ZFN and donor into host cells,less studies have investigated the relationship between the homologous arm length with the efficiency of ZFN induced homologous recombination.Here,we constructed a pair of ZFN plasmids targeting to EGFP and verified its cutting activity.Then we designed a series of donors with dif-ferent lengths of homologous arms.By introducing individual donor with ZFN into CHO cells harboring a frame-shift GFP gene,we measured the homologous recombination efficiencies through the flow cytometric analysis.We found that a 50 bp short homology arm was capable to support ZFN-mediated homologous re-combination.Increasing the length of the homologous arms could improve the efficiency of ZFN-mediated homologous recombination.A dramatic improvement (104-fold higher than traditional method)requires a homology arm longer than 1 000 bp.
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