Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.
Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.
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DOI：
10.1093/hmg/ddv409
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发表时间：
2015-12-15
影响因子：
3.5
通讯作者：
Porter AC
中科院分区：
文献类型：
--
作者：
Gravells P;Ahrabi S;Vangala RK;Tomita K;Brash JT;Brustle LA;Chung C;Hong JM;Kaloudi A;Humphrey TC;Porter AC
了解染色体双链断裂修复（DSBR）的机制有助于深入了解基因组不稳定性、肿瘤发生和基因组工程，包括疾病基因校正。DSBR的研究利用稀有切割内切酶切割外源报告基因构建物整合到基因组中。已经开发了多个报告结构来检测各种DSBR通路。在这里，我们使用一个单一的内源性报告基因，编码次黄嘌呤磷酸核糖基转移酶（HPRT）的x染色体疾病基因，监测了被I-SceI或CRISPR/Cas9核酸酶切割后三种DSBR通路的相对利用。对于I-SceI，我们估计准确或诱变的非同源末端连接和同源重组基因校正的频率分别为4.1、1.5和0.16%。出乎意料的是，I-SceI和Cas9诱导的DSBR谱明显不同。此外，使用i - scii敏感的HPRT迷你基因，我们表明，当使用长双链DNA时，基因校正比单链或双链寡核苷酸更有效。最后，利用内源性HPRT和外源性报告基因，我们验证了新的细胞周期相特异性I-SceI衍生物，用于研究DSBR的细胞周期变化。使用这些新方法获得的结果为基因校正模板设计和单个内源性疾病基因上多个DSBR通路之间的关系提供了新的见解。
Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.