Sangamo BioSciences, 501 Canal Bvld, Richmond, CA 94804, USA.
Nucleic Acids Res. 2010 Aug;38(15):e152. doi: 10.1093/nar/gkq512. Epub 2010 Jun 8.
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 approximately 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.
我们之前展示了通过同源定向 DNA 修复途径介导的高频、靶向 DNA 添加。该方法使用锌指核酸酶（ZFN）在特定位点创建双链断裂（DSB），从而促进从外源供体分子将遗传信息复制到染色体中。此类供体通常包含两个大约 750 bp 的染色体序列区域，是同源定向 DNA 修复所必需的。在这里，我们证明了易于生成的线性供体，其具有极短的（50 bp）同源区域，可将转基因整合到 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 产生的单链 5'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。通过依赖于和受供体 DNA 上互补 5'突出端指导的过程，超过 10%的所有染色体直接掺入这种外源性 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 对同时作用产生的大片段缺失中来扩展这种非同源末端连接（NHEJ）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添了多功能性。
