Biased genome editing using the local accumulation of DSB repair molecules system.
Biased genome editing using the local accumulation of DSB repair molecules system.
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DOI：
10.1038/s41467-018-05773-6
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发表时间：
2018-08-16
影响因子：
16.6
通讯作者：
Yamamoto T
中科院分区：
文献类型：
--
作者：
Nakade S;Mochida K;Kunii A;Nakamae K;Aida T;Tanaka K;Sakamoto N;Sakuma T;Yamamoto T
选择性基因组编辑，如基因敲入，最近已经通过施用特定DNA双链断裂（DSB）修复途径的化学增强剂或抑制剂以及途径特异性基因的过表达来实现。在本研究中，我们试图通过使用DSB修复分子的局部积累（LoAD）系统来进一步提高效率以确保稳健的基因敲入。我们通过遗传筛选将CtIP鉴定为微同源介导的末端连接（MMEJ）修复的强增强子，并显示CtIP加载的敲入增强效应。下一代测序表明，CtIP加载大大增加了MMEJ介导的整合频率。无选择，同时三重基因敲入也实现了与CtIP-加载策略。此外，通过替换LoADing分子和靶向策略，该系统可以应用于其他特定的基因组工程目的，例如引入更长的缺失以进行基因破坏，独立地引入多个突变而没有染色体缺失，以及有效地掺入单链寡脱氧核苷酸供体。使用CRISPR的基因组编辑可以通过操纵DNA双链断裂修复途径来增强。在这里，作者证明了修复分子的局部积累，将修复转移到微同源介导的末端连接。
Selective genome editing such as gene knock-in has recently been achieved by administration of chemical enhancer or inhibitor of particular DNA double-strand break (DSB) repair pathways, as well as overexpression of pathway-specific genes. In this study, we attempt to enhance the efficiency further to secure robust gene knock-ins, by using the local accumulation of DSB repair molecules (LoAD) system. We identify CtIP as a strong enhancer of microhomology-mediated end-joining (MMEJ) repair by genetic screening, and show the knock-in-enhancing effect of CtIP LoADing. Next-generation sequencing reveals that CtIP LoADing highly increases the frequency of MMEJ-mediated integration. Selection-free, simultaneous triple gene knock-ins are also achieved with the CtIP-LoADing strategy. Moreover, by replacing the LoADing molecules and targeting strategies, this system can be applied for other specific genome engineering purposes, such as introducing longer deletions for gene disruption, independently introducing multiple mutations without chromosomal deletion, and efficiently incorporating a single-stranded oligodeoxynucleotide donor. Genome editing using CRISPR can be enhanced by manipulating DNA double-strand break repair pathways. Here the authors demonstrate LoAD, local accumulation of repair molecules, which shifts repair to microhomology-mediated end-joining.
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影响因子：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
影响因子：
4.6
作者：
Sakuma T;Nishikawa A;Kume S;Chayama K;Yamamoto T
通讯作者：
Yamamoto T
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：
4.6
作者：
Hisano Y;Sakuma T;Nakade S;Ohga R;Ota S;Okamoto H;Yamamoto T;Kawahara A
通讯作者：
Kawahara A
DOI：
10.1083/jcb.201401146
发表时间：
2014-09-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Barton O;Naumann SC;Diemer-Biehs R;Künzel J;Steinlage M;Conrad S;Makharashvili N;Wang J;Feng L;Lopez BS;Paull TT;Chen J;Jeggo PA;Löbrich M
通讯作者：
Löbrich M