鋅指核酸酶

鋅指核酸酶（英語：Zinc-finger nucleases, ZFNs），是人工改造的限制酶，通過融合鋅指結構的結合DNA結構域和分解DNA結構域而成。可通過基因工程改造鋅指結構域使鋅指核酸酶針對複雜基因組裡的特定DNA序列。藉助內源DNA的修復機制，鋅指核酸酶可以精確改變高等動物的基因組。類似的技術還有類轉錄活化因子核酸酶。

結構域

鋅指核酸酶是一種特定於位點的核酸限制內切酶

功能：設計在特定的位置來結合併分解DNA，含有兩個蛋白質結構域。

第一個結構域是DNA結合結構域，其由真核轉錄因子並包含鋅指所構成；第二個結構域是核酸酶結構域，它由FokI限制酶構成，並負責DNA的催化分解。

分解DNA結構域

A pair of two ZFNs with three zinc fingers each are shown introducing a double-strand break. Subsequent to this, the double strand break is being repaired through either homologous recombination or non-homologous end joining.^[1]

type IIs 核酸內切酶 FokI 的非特異分解域經常被用做 ZFNs 的分解域，^[2] 這個分解域需要二聚化來分解DNA。^[3] 因此一對ZFNs只能用於定位非回文的DNA位點。標準的ZFNs每個域的碳端都融合了分離域。為了讓兩個分離域二聚化並分解DNA,兩個單獨的ZFNs需要結合在不同的DNA鏈上並且他們的碳端相隔一定的距離。在鋅指域和分解域之間最常用的連結序列要求每個結合位點的5'端分隔5到7個bp。^[4]

幾個不同的蛋白質工程技術已經用來提高用於ZFNs核酸酶域的活性和特異性。直接進化被用來產生FokI的多樣性以增強被作者稱為"Sharkey"的^[5] 分解活性。通過修改二聚化的接口以便只有目的異二聚體類型有活性，基於結構的設計也被用來提高FokI的分解特異性。^[6]^[7]^[8]^[9]

結合DNA結構域

應用

去除一個等位基因

等位基因編輯

基因治療

潛在問題

脫靶分解

免疫原性

前景

鋅指切口酶

鋅指核酸酶治療HIV

鋅指結合

核酸酶二聚化和分離

CCR5突變的介紹

局限性

治療措施

參見

參考文獻

^ 引用錯誤：沒有為名為genomeengineering的參考文獻提供內容
^ Kim, YG; Cha, J.; Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 1996, 93 (3): 1156–60 [2014-12-05]. Bibcode:1996PNAS...93.1156K. PMC 40048  . PMID 8577732. doi:10.1073/pnas.93.3.1156. （原始內容存檔於2015-01-28）.
^ Bitinaite, J.; D. A. Wah, Aggarwal, A. K., Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci USA. 1998, 95 (18): 10570–5 [2014-12-05]. Bibcode:1998PNAS...9510570B. PMC 27935  . PMID 9724744. doi:10.1073/pnas.95.18.10570. （原始內容存檔於2005-04-24）.
^ Cathomen T, Joung JK. Zinc-finger nucleases: the next generation emerges. Mol. Ther. July 2008, 16 (7): 1200–7 [2014-12-05]. PMID 18545224. doi:10.1038/mt.2008.114. （原始內容存檔於2015-02-09）.
^ Guo, Jing; Gaj, Thomas; Barbas, Carlos F. Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases. Journal of Molecular Biology (Elsevier BV). 2010, 400 (1): 96–107. ISSN 0022-2836. doi:10.1016/j.jmb.2010.04.060.
^ Szczepek, Michal; Brondani, Vincent; Büchel, Janine; Serrano, Luis; Segal, David J; Cathomen, Toni. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nature Biotechnology (Springer Nature). 2007, 25 (7): 786–793. ISSN 1087-0156. doi:10.1038/nbt1317.
^ Miller, Jeffrey C; Holmes, Michael C; Wang, Jianbin; Guschin, Dmitry Y; Lee, Ya-Li; Rupniewski, Igor; Beausejour, Christian M; Waite, Adam J; Wang, Nathaniel S; Kim, Kenneth A; Gregory, Philip D; Pabo, Carl O; Rebar, Edward J. An improved zinc-finger nuclease architecture for highly specific genome editing. Nature Biotechnology (Springer Nature). 2007, 25 (7): 778–785. ISSN 1087-0156. doi:10.1038/nbt1319.
^ Doyon, Yannick; Vo, Thuy D; Mendel, Matthew C; Greenberg, Shon G; Wang, Jianbin; Xia, Danny F; Miller, Jeffrey C; Urnov, Fyodor D; Gregory, Philip D; Holmes, Michael C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nature Methods (Springer Nature). 2010-12-05, 8 (1): 74–79. ISSN 1548-7091. doi:10.1038/nmeth.1539.
^ Ramalingam, Sivaprakash; Kandavelou, Karthikeyan; Rajenderan, Raja; Chandrasegaran, Srinivasan. Creating Designed Zinc-Finger Nucleases with Minimal Cytotoxicity. Journal of Molecular Biology (Elsevier BV). 2011, 405 (3): 630–641. ISSN 0022-2836. doi:10.1016/j.jmb.2010.10.043.

延伸閱讀

Mandell JG, Barbas CF. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. July 2006, 34 (Web Server issue): W516–23 [2009-09-25]. PMC 1538883  . PMID 16845061. doi:10.1093/nar/gkl209. （原始內容存檔於2019-07-10）.
Porteus MH, Carroll D. Gene targeting using zinc finger nucleases. Nat. Biotechnol. August 2005, 23 (8): 967–73. PMID 16082368. doi:10.1038/nbt1125.
Doyon Y, McCammon JM, Miller JC; et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. June 2008, 26 (6): 702–8. PMC 2674762  . PMID 18500334. doi:10.1038/nbt1409.
Meng X, Noyes MB, Zhu LJ, Lawson ND, Wolfe SA. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. June 2008, 26 (6): 695–701. PMC 2502069  . PMID 18500337. doi:10.1038/nbt1398.

外部連結

Zinc finger selector （頁面存檔備份，存於網際網路檔案館）
Zinc Finger Consortium website （頁面存檔備份，存於網際網路檔案館）
Zinc Finger Consortium materials from Addgene （頁面存檔備份，存於網際網路檔案館）
A commercial supplier of ZFNs （頁面存檔備份，存於網際網路檔案館）

[genomeengineering-1] 引用錯誤：沒有為名為genomeengineering的參考文獻提供內容

[kim1996-2] Kim, YG; Cha, J.; Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 1996, 93 (3): 1156–60 [2014-12-05]. Bibcode:1996PNAS...93.1156K. PMC 40048  . PMID 8577732. doi:10.1073/pnas.93.3.1156. （原始內容存檔於2015-01-28）.

[bitinaite1998-3] Bitinaite, J.; D. A. Wah, Aggarwal, A. K., Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci USA. 1998, 95 (18): 10570–5 [2014-12-05]. Bibcode:1998PNAS...9510570B. PMC 27935  . PMID 9724744. doi:10.1073/pnas.95.18.10570. （原始內容存檔於2005-04-24）.

[4] Cathomen T, Joung JK. Zinc-finger nucleases: the next generation emerges. Mol. Ther. July 2008, 16 (7): 1200–7 [2014-12-05]. PMID 18545224. doi:10.1038/mt.2008.114. （原始內容存檔於2015-02-09）.

[Guo2010-5] Guo, Jing; Gaj, Thomas; Barbas, Carlos F. Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases. Journal of Molecular Biology (Elsevier BV). 2010, 400 (1): 96–107. ISSN 0022-2836. doi:10.1016/j.jmb.2010.04.060.

[Szczepek2007-6] Szczepek, Michal; Brondani, Vincent; Büchel, Janine; Serrano, Luis; Segal, David J; Cathomen, Toni. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nature Biotechnology (Springer Nature). 2007, 25 (7): 786–793. ISSN 1087-0156. doi:10.1038/nbt1317.

[Miller2007-7] Miller, Jeffrey C; Holmes, Michael C; Wang, Jianbin; Guschin, Dmitry Y; Lee, Ya-Li; Rupniewski, Igor; Beausejour, Christian M; Waite, Adam J; Wang, Nathaniel S; Kim, Kenneth A; Gregory, Philip D; Pabo, Carl O; Rebar, Edward J. An improved zinc-finger nuclease architecture for highly specific genome editing. Nature Biotechnology (Springer Nature). 2007, 25 (7): 778–785. ISSN 1087-0156. doi:10.1038/nbt1319.

[Doyon2010-8] Doyon, Yannick; Vo, Thuy D; Mendel, Matthew C; Greenberg, Shon G; Wang, Jianbin; Xia, Danny F; Miller, Jeffrey C; Urnov, Fyodor D; Gregory, Philip D; Holmes, Michael C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nature Methods (Springer Nature). 2010-12-05, 8 (1): 74–79. ISSN 1548-7091. doi:10.1038/nmeth.1539.

[Ramalingam2010-9] Ramalingam, Sivaprakash; Kandavelou, Karthikeyan; Rajenderan, Raja; Chandrasegaran, Srinivasan. Creating Designed Zinc-Finger Nucleases with Minimal Cytotoxicity. Journal of Molecular Biology (Elsevier BV). 2011, 405 (3): 630–641. ISSN 0022-2836. doi:10.1016/j.jmb.2010.10.043.

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