TALENs新型模块组装法及活性鉴定方法

doi:10.13560/j.cnki.biotech.bull.1985.2016.01.035

生物技术通报 ›› 2016, Vol. 32 ›› Issue (1): 220-228.doi: 10.13560/j.cnki.biotech.bull.1985.2016.01.035

TALENs新型模块组装法及活性鉴定方法

高敬^{1, 2}, 魏迪^{1, 2}, 池振奋³, 张癸荣^{1, 2}, 张万明¹, 聂凌云^{1, 2}

1.河北北方学院, 张家口 075000;
2.中国人民解放军总后勤部卫生部药品仪器检验所, 北京 100166;
3.中国科学院北京基因组研究所, 北京 100101

收稿日期:2015-03-04 出版日期:2016-01-09 发布日期:2016-01-22
作者简介:高敬, 女, 硕士研究生, 研究方向：遗传学研究新技术与新方法;E-mail：gaojing201201@126.com
基金资助:
国家自然科学基金面上项目(31371346)

A Novel Method of Unit Assembly and Activity Assay for Transcription Activator-like Effector Nucleases

GAO Jing^{1, 2}, WEI Di^{1, 2}, CHI Zhen-fen³, ZHANG Gui-rong^{1, 2}, ZHANG Wan-ming¹, NIE Ling-yun^{1, 2}

1. Hebei North University, Zhangjiakou 075000;
2. Institute for Drug and Instrument Control, Health Department, the General Logistics Department of People’s Liberation Army, Beijing 100166;
3. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101

Received:2015-03-04 Published:2016-01-09 Online:2016-01-22

摘要/Abstract

摘要： 旨在提供一种新型的转录激活样效应子核酸酶(Transcription activator-like effector nucleases, TALENs)模块组装法(Unit assembly, UA)及活性鉴定方法。利用TALE-NT 2.0在线工具在线粒体DNA(Mitochondrial DNA, mtDNA)设计TALENs识别位点和剪切位点, 借助pEGFP-N1质粒将该段靶序列随机整合于HEK293F核基因组中, 构建HEK293F-T1细胞系, 用于TALENs活性鉴定。依据转录激活样效应子(Transcription activator-like effectors, TALEs)自然单元模块序列, 设计出一种新型的人工TALEs偏单元;根据TALENs识别位点, 选取相应一单元模块, 利用UA法组装;并设计出含有相应双酶切位点的TALEs串联模块序列和TALENs载体序列, 定向连接后瞬时转染HEK293F-T1细胞系。结果显示, 新型模块组装法定向组装TALEs模块, 克隆效率高且瞬时转染后可看到清晰的套峰。结果表明, 新型模块组装方法提高了TALENs构建过程中的克隆效率, 并且不受末位0.5模块的RVD(repeat-variable diresidue)限制, 增加了靶序列设计的灵活性。

关键词: 转录激活样效应子核酸酶, 模块组装法, 线粒体DNA, HEK293F-T1

Abstract: A novel method of unit assembly(UA)and activity assay based on transcription activator-like effector nucleases(TALENs)are described. An online tool TALE-NT 2.0 was used to design recognition and splice sites on mitochondrial DNA(mtDNA)of TALENs. By means of pEGFP-N1 plasmid, the segments of target sequence were randomly integrated in the nuclear genome of HEK293F. The constructed cell line HEK293F-T1 was for activity assay of TALENs. Based on transcription activator-like effector(TALE)natural repeats, a new type of artificial TALEs single-unit repeats were designed. According to the recognition sites of TALENs, appropriate single-unit repeats were selected and assembled by UA. TALEs series unit sequence containing the corresponding double enzyme cutting sites and TALENs vector sequence were designed, which was directionally ligated and transiently transfected into HEK293F-T1 cell line. The results showed that the TALES units were directionally assembled by new method, and it had a high cloning efficiency;moreover, the nested peaks clearly appeared after transient transfection. In conclusion, the novel method of unit assembly improves cloning efficiency of constructing TALLENs, even is not limited by repeat-variable residue(RVD)in the last 0.5 unit, and increases the flexibility of designing the target sequences.

Key words: transcription activator-like effector nucleases, unit assembly, mitochondrial DNA, HEK293F-T1

高敬, 魏迪, 池振奋, 张癸荣, 张万明, 聂凌云. TALENs新型模块组装法及活性鉴定方法[J]. 生物技术通报, 2016, 32(1): 220-228.

GAO Jing, WEI Di, CHI Zhen-fen, ZHANG Gui-rong, ZHANG Wan-ming, NIE Ling-yun. A Novel Method of Unit Assembly and Activity Assay for Transcription Activator-like Effector Nucleases[J]. Biotechnology Bulletin, 2016, 32(1): 220-228.

参考文献

［1］ Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering［J］. Trends in Biotechnology, 2013, 31(7)：397-405.
［2］ Moscou MJ, Bogdanove AJ. A Simple Cipher Governs DNA Recognition by TAL Effectors［J］. Science, 2009, 326(5959)：1501.
［3］ Chen L, Tang L, Xiang H, et al. Advances in genome editing technology and its promising application in evolutionary and ecological studies［J］. GigaScience, 2014, 3：24.
［4］ Christian M, Cermak T, Doyle EL, et al. Targeting DNA double-strand breaks with TAL effector nucleases［J］. Genetics, 2010, 186(2)：757-761.
［5］ Boch J, Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors［J］. Science, 2009, 326(5959)：1509-1512.
［6］ Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors：discovery and function［J］. Annual Review of Phytopathology, 2010, 48：419-436.
［7］ Huang P, Xiao A, Zhou MG, et al. Heritable gene targeting in zebrafish using customized TALENs［J］. Nature Biotechnology, 2011, 29(8)：699-700.
［8］ Miller JC, Tan S, Qiao G, et al. A TALE nuclease architecture for efficient genome editing［J］. Nature Biotechnology, 2011, 29(2)：143-148.
［9］ Szurek B, Rossier O, Hause G, et al. Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell［J］. Mol Microbiol, 2002, 46(1)：13-23.
［10］ Mussolino C, Morbitzer R, Lutge F, et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity［J］. Nucleic Acids Research, 2011, 39(21)：9283-9293.
［11］ Li T, Liu B, Spalding MH, et al. High-efficiency TALEN-based gene editing produces disease-resistant rice［J］. Nature Biotechnology, 2012, 30(5)：390-392.
［12］ Bacman SR, Williams SL, Pinto M, et al. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs［J］. Nature Medicine, 2013, 19(9)：1111-1113.
［13］ Cermak T, Doyle EL, Christian M, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting［J］. Nucleic Acids Research, 2011, 39(12)：e82.
［14］ Weber E, Gruetzner R, Werner S, et al. Assembly of designer TAL effectors by Golden Gate cloning［J］. PloS One, 2011, 6(5)：e19722.
［15］ Zhang F, Cong L, Lodato S, et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription［J］. Nature Biotechnology, 2011, 29(2)：149-153.
［16］ Sanjana NE, Cong L, Zhou Y, et al. A transcription activator-like effector toolbox for genome engineering［J］. Nature Protocols, 2012, 7(1)：171-192.
［17］ Reyon D, Tsai SQ, Khayter C, et al. FLASH assembly of TALENs for high-throughput genome editing［J］. Nature Biotechnology, 2012, 30(5)：460-465.
［18］ Reyon D, Maeder ML, Khayter C, et al. Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly［J］. Current Protocols in Molecular Biology, 2013, Chapter 12：Unit 12.6.
［19］ Schmid-Burgk JL, Schmidt T, Kaiser V, et al. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes［J］. Nature Biotechnology, 2013, 31(1)：76-81.
［20］ Briggs AW, Rios X, Chari R, et al. Iterative capped assembly：rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers［J］. Nucleic Acids Research, 2012, 40(15)：e117.
［21］ Li L, Piatek MJ, Atef A, et al. Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification［J］. Plant Mol Biol, 2012, 78(4-5)：407-416.
［22］ Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs［J］. Nature Biotechnology, 2011, 29(8)：697-698.
［23］ Larsen NB, Rasmussen M, Rasmussen L J. Nuclear and mitochond-rial DNA repair：similar pathways?［J］. Mitochondrion, 2005, 5(2)：89-108.
［24］沈延, 肖安, 黄鹏, 等. 类转录激活因子效应物核酸酶(TALEN)介导的基因组定点修饰技术［J］. 遗传, 2013, 35(4)：395-409.

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TALENs新型模块组装法及活性鉴定方法

A Novel Method of Unit Assembly and Activity Assay for Transcription Activator-like Effector Nucleases

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