The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.
The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.
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DOI：
10.1371/journal.pone.0096483
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kmiec EB
中科院分区：
文献类型：
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作者：
Rivera-Torres N;Strouse B;Bialk P;Niamat RA;Kmiec EB
随着最近的技术进步使得DNA能够在人类基因组的特定位点进行切割，现在有可能扭转先天性错误，从而纠正突变，其水平可能对临床环境产生影响。我们一直在开发基因编辑，使用单链 DNA 寡核苷酸 (ssODN) 作为指导位点特异性单碱基变化的工具。该技术的成功应用已在从细菌到人类（ES 和体细胞）细胞的许多系统中得到证实。虽然基因编辑的频率差异很大，但通常处于无法临床应用的水平。因此，已知许多刺激因素（例如双链断裂）会显着提高频率。其中大部分结果是使用经过验证的 HCT116 哺乳动物细胞模型系统发现的，该系统可以提供可靠的遗传和生化读数。在这里，我们将执行特定 ds DNA 断裂的 TAL 效应核酸酶 (TALEN) 与 ssODN 结合在一起，ssODN 专门设计用于修复集成的单拷贝 eGFP 基因中的错义突变。我们发现，相对于突变体碱基的近端切割是实现高频率编辑的关键。还观察到校正的方向性，目标碱基上游的 TALEN 活性比下游活性更有效地促进基因编辑。我们还发现，进入 S 期的细胞更适合组合基因编辑活动。因此，我们确定了基因编辑的新颖方面，这将有助于设计更有效的基因组修饰和自然基因基因治疗方案。
With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.
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影响因子：
5.1
作者：
Igoucheva, O;Alexeev, V;Yoon, K
通讯作者：
Yoon, K
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
DOI：
10.1038/mtna.2012.51
发表时间：
2012-12-04
期刊：
Molecular therapy. Nucleic acids
影响因子：
--
作者：
通讯作者：
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影响因子：
23.9
作者：
Ding, Qiurong;Lee, Youn-Kyoung;Schaefer, Esperance A. K.;Peters, Derek T.;Veres, Adrian;Kim, Kevin;Kuperwasser, Nicolas;Motola, Daniel L.;Meissner, Torsten B.;Hendriks, William T.;Trevisan, Marta;Gupta, Rajat M.;Moisan, Annie;Banks, Eric;Friesen, Max;Schinzel, Robert T.;Xia, Fang;Tang, Alexander;Xia, Yulei;Figueroa, Emmanuel;Wann, Amy;Ahfeldt, Tim;Daheron, Laurence;Zhang, Feng;Rubin, Lee L.;Peng, Lee F.;Chung, Raymond T.;Musunuru, Kiran;Cowan, Chad A.
通讯作者：
Cowan, Chad A.
影响因子：
64.8
作者：
通讯作者：
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