In vivo genome editing using a high-efficiency TALEN system.
In vivo genome editing using a high-efficiency TALEN system.
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作者：
斑马鱼（Danio rerio）越来越多地被用于研究基础脊椎动物生物学和人类疾病，使用丰富的体内遗传和分子工具。然而，不能以靶向方式容易地修饰基因组一直是该领域的瓶颈。在这里，我们表明，人工转录激活因子样效应核酸酶（TALEN）的改进为靶向斑马鱼基因组编辑和功能基因组应用提供了一种强大的新方法。使用GoldyTALEN修饰的支架和斑马鱼递送系统，我们表明这种增强的TALEN工具包在体细胞和种系组织中诱导基因座特异性DNA断裂方面表现出高效率。在一些基因座，这种功效接近100%，包括体细胞组织中的双等位基因转换，其模拟使用基于吗啉代（MO）的靶向基因敲除所见的表型。通过这种更新的TALEN系统，我们成功地使用单链DNA（ssDNA）寡核苷酸（oligos）通过同源定向修复（HDR）精确地修饰斑马鱼基因组中预定义位置的序列，包括将定制设计的EcoRV位点和修饰的loxP（mloxP）序列引入体内体组织。我们进一步显示了EcoRV和mloxP工程化染色体的成功种系传递。这种组合方法提供了模拟遗传变异以及产生靶向条件等位基因的潜力。
The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease using a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show this enhanced TALEN toolkit demonstrates a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino (MO)-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA (ssDNA) oligonucleotides (oligos) to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair (HDR), including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
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影响因子：
2.7
作者：
Blum, Yannick;Belting, Heinz-Georg;Affolter, Markus
通讯作者：
Affolter, Markus
DOI：
10.1073/pnas.1019533108
发表时间：
2011-02-08
影响因子：
11.1
作者：
Mahfouz, Magdy M.;Li, Lixin;Zhu, Jian-Kang
通讯作者：
Zhu, Jian-Kang
影响因子：
4.6
作者：
Bedell, Victoria M.;Person, Anthony D.;Ekker, Stephen C.
通讯作者：
Ekker, Stephen C.
影响因子：
14.9
作者：
Cade L;Reyon D;Hwang WY;Tsai SQ;Patel S;Khayter C;Joung JK;Sander JD;Peterson RT;Yeh JR
通讯作者：
Yeh JR
影响因子：
4.6
作者：
Wang, Ying;Kaiser, Mark S.;Essner, Jeffrey J.
通讯作者：
Essner, Jeffrey J.