Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease

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• 作者：Kazuki  ; Shinomiya ; Tomoaki  ; Mori ; Yasuhiro  ; Aoyama ; Takashi  ; Sera  ; ;   ; ^{sera@sbchem.kyoto-u.ac.jp}
• 关键词：Sandwiched zinc finger nuclease ; Single-chain FokI dimer ; Zinc finger protein ; DNA cloning ; Meganuclease
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2011
• 出版时间：4 November 2011
• 年：2011
• 卷：414
• 期：4
• 页码：733-736
• 全文大小：472 K

文摘

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1 h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (?6-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.