A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.
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DOI：
10.1093/nar/gkr597
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发表时间：
2011-11
影响因子：
14.9
通讯作者：
Cathomen T
中科院分区：
文献类型：
--
作者：
Mussolino C;Morbitzer R;Lütge F;Dannemann N;Lahaye T;Cathomen T
序列特异性核酸酶代表用于精确基因组工程的有价值的工具。传统上，锌指核酸酶（ZFN）和大范围核酸酶已被用于特异性编辑复杂的基因组。最近，来自细菌病原体黄单胞菌属的转录激活因子样效应子（TALE）的DNA结合结构域已被利用来将核酸酶结构域引导至所需的基因组基因座。在这项研究中，我们测试了一组基于TALE蛋白AvrBs4的截短变体，以鉴定具有高DNA切割活性的TALE核酸酶（TALEN）。在体外和细胞报告基因测定中确定了有效DNA切割的最有利参数。设计TALEN以破坏EGFP标记基因和人基因座CCR5和IL2RG。在高达45%的转染细胞中实现了基因编辑。与ZFN的并排比较显示，TALEN具有相似的基因破坏活性，但显着降低了核酸酶相关的细胞毒性。此外，与相应的ZFN相比，CCR5特异性TALEN在CCR2基因座处仅显示最小的脱靶活性，表明TALEN平台能够设计具有单核苷酸特异性的核酸酶。高核酸酶活性与降低的细胞毒性和简单的设计过程相结合，标志着TALEN成为复杂基因组靶向修饰的关键技术平台。
Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.
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影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
14.9
作者：
Li T;Huang S;Jiang WZ;Wright D;Spalding MH;Weeks DP;Yang B
通讯作者：
Yang B
影响因子：
12.4
作者：
Alwin, S;Gere, MB;Cathomen, T
通讯作者：
Cathomen, T
DOI：
10.1007/978-1-60761-753-2_14
发表时间：
2010-01-01
期刊：
ENGINEERED ZINC FINGER PROTEINS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Cornu, Tatjana I.;Cathomen, Toni
通讯作者：
Cathomen, Toni
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla