TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus
TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus
复制标题
TALE 切口酶介导人类多拷贝核糖体 DNA 基因座的高效靶向转基因整合
DOI：
10.1016/j.bbrc.2014.02.099
复制
发表时间：
2014-03-28
影响因子：
3.1
通讯作者：
Liang, Desheng
中科院分区：
文献类型：
--
作者：
Wu, Yong;Gao, Tieli;Liang, Desheng
虽然靶向基因的添加可以通过由锌指核酸酶（ZFN）或TALE核酸酶（TALEN）产生的DNA双链断裂（DSB）来显著刺激，但DSB对人类细胞确实具有致突变性和毒性。作为一种折衷的解决方案，DNA单链断裂（SSB）或切口已被报道介导高效率的基因添加，但随机诱变显着减少。我们先前证明了在人类多拷贝核糖体DNA（rDNA）位点有效的靶向基因添加，这是具有治疗潜力的转基因的基因组安全港。为了通过使用TALEN提高转基因整合效率，同时降低DSB的细胞毒性，我们产生了靶向该多拷贝基因座的TALEN和TALE切口酶（TALENickases）。构建了可以在rDNA基因座处整合GFP盒的靶向载体，并与TALEN或TALENickases共转染。尽管在转染后的最初几天内使用TALEN的GFP阳性细胞的分数大于使用TALENickases的GFP阳性细胞的分数，但在连续培养后其降低至低于使用TALENickases的GFP阳性细胞的水平。我们的研究结果表明，TALENickases在多拷贝rDNA基因座上比TALEN对应物更有效，尽管早期使用ZFN和ZFNickases靶向单拷贝基因座的研究显示了相反的结果。此外，TALENickases在药物选择后以高达0.62%的频率介导5.4kb片段在HT1080细胞中的靶向整合，表明它们在靶向基因修饰中的潜在应用不限于rDNA位点。(C)2014爱思唯尔公司All rights reserved.
Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus. (C) 2014 Elsevier Inc. All rights reserved.