Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.
Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.
复制标题
DOI：
10.1093/nar/gkq512
复制
发表时间：
2010-08
影响因子：
14.9
通讯作者：
Cost GJ
中科院分区：
文献类型：
--
作者：
Orlando SJ;Santiago Y;DeKelver RC;Freyvert Y;Boydston EA;Moehle EA;Choi VM;Gopalan SM;Lou JF;Li J;Miller JC;Holmes MC;Gregory PD;Urnov FD;Cost GJ
我们以前证明了高频率，有针对性的DNA添加介导的同源性指导的DNA修复途径。该方法使用锌指核酸酶（ZFN）来产生位点特异性双链断裂（DSB），其促进遗传信息从外源供体分子复制到染色体中。这样的供体通常含有同源定向DNA修复所需的染色体序列的两个约750 bp区域。在这里，我们证明了具有极短（50 bp）同源区域的容易产生的线性供体驱动转基因整合到5-10%的染色体中。此外，我们测量了ZFN切割产生的突出端，发现具有与ZFN产生的突出端互补的单链5′突出端的寡核苷酸供体在体内有效地连接到DSB。超过10%的染色体通过依赖于供体DNA上的互补5′突出端并由其引导的过程直接掺入这种外源DNA。最后，我们通过将包含重组酶位点的供体DNA直接插入由两个单独的ZFN对的同时作用产生的大缺失中来扩展这种基于非同源末端连接（NHEJ）的技术。多达50%的缺失包含供体插入。通过NHEJ的靶向DNA添加补充了我们的同源定向靶向整合方法，为哺乳动物基因组的操作增加了多功能性。
We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two ∼750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5–10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5′ overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5′ overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.
登录
查看更多内容
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：
7
作者：
Lee, Hyung Joo;Kim, Eunji;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
14.9
作者：
ARATANI, Y;OKAZAKI, R;KOYAMA, H
通讯作者：
KOYAMA, H
影响因子：
3.8
作者：
Cost, Gregory J.;Freyvert, Yevgeniy;Gregory, Philip D.
通讯作者：
Gregory, Philip D.
影响因子：
64.8
作者：
CHOO, Y;SANCHEZGARCIA, I;KLUG, A
通讯作者：
KLUG, A