锌指核酸酶

锌指核酸酶（英语：Zinc-finger nucleases, ZFNs），是人工改造的限制酶，通过融合锌指结构的结合DNA结构域和分解DNA结构域而成。可通过基因工程改造锌指结构域使锌指核酸酶针对复杂基因组里的特定DNA序列。借助内源DNA的修复机制，锌指核酸酶可以精确改变高等动物的基因组。类似的技术还有类转录活化因子核酸酶。

结构域[编辑]

锌指核酸酶是一种特定于位点的核酸限制内切酶

功能：设计在特定的位置来结合并分解DNA，含有两个蛋白质结构域。

第一个结构域是DNA结合结构域，其由真核转录因子并包含锌指所构成；第二个结构域是核酸酶结构域，它由FokI限制酶构成，并负责DNA的催化分解。

分解DNA结构域[编辑]

type IIs 核酸内切酶 FokI 的非特异分解域经常被用做 ZFNs 的分解域，^[2] 这个分解域需要二聚化来分解DNA。^[3] 因此一对ZFNs只能用于定位非回文的DNA位点。标准的ZFNs每个域的碳端都融合了分离域。为了让两个分离域二聚化并分解DNA,两个单独的ZFNs需要结合在不同的DNA链上并且他们的碳端相隔一定的距离。在锌指域和分解域之间最常用的链接序列要求每个结合位点的5'端分隔5到7个bp。^[4]

几个不同的蛋白质工程技术已经用来提高用于ZFNs核酸酶域的活性和特异性。直接进化被用来产生FokI的多样性以增强被作者称为"Sharkey"的^[5] 分解活性。通过修改二聚化的接口以便只有目的异二聚体类型有活性，基于结构的设计也被用来提高FokI的分解特异性。^[6]^[7]^[8]^[9]

结合DNA结构域[编辑]

应用[编辑]

去除一个等位基因[编辑]

等位基因编辑[编辑]

基因治疗[编辑]

潜在问题[编辑]

脱靶分解[编辑]

免疫原性[编辑]

前景[编辑]

锌指切口酶[编辑]

锌指核酸酶治疗HIV[编辑]

锌指结合[编辑]

核酸酶二聚化和分离[编辑]

CCR5突变的介绍[编辑]

局限性[编辑]

治疗措施[编辑]

参见[编辑]

参考文献[编辑]

^ 引证错误：没有为名为genomeengineering的参考文献提供内容
^ 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. （原始内容存档于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. （原始内容存档于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. （原始内容存档于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.

延伸阅读[编辑]

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. （原始内容存档于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.

外部链接[编辑]

Zinc finger selector （页面存档备份，存于互联网档案馆）
Zinc Finger Consortium website （页面存档备份，存于互联网档案馆）
Zinc Finger Consortium materials from Addgene （页面存档备份，存于互联网档案馆）
A commercial supplier of ZFNs （页面存档备份，存于互联网档案馆）

[genomeengineering-1] 引证错误：没有为名为genomeengineering的参考文献提供内容

[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. （原始内容存档于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. （原始内容存档于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. （原始内容存档于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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