Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair.
Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair.
复制标题
DOI：
10.1101/gr.226027.117
复制
发表时间：
2018-03
期刊：
影响因子：
7
通讯作者：
Nakada S
中科院分区：
文献类型：
--
作者：
Nakajima K;Zhou Y;Tomita A;Hirade Y;Gurumurthy CB;Nakada S
CRISPR/Cas9在靶位点产生DNA双链断裂（DSB），是与供体DNA模板共同递送时编辑基因组的强大工具。然而，DSB是最有害的DNA损伤类型，通常通过诱变性非同源末端连接导致非预期的核苷酸插入/缺失（indel）。我们开发了一种不产生DSB的精确基因编辑策略。我们表明，使用Cas 9D 10A切口酶的靶基因和供体质粒（SNGD）中的单个切口的组合通过基因编辑促进了有效的核苷酸取代。单独切割靶基因并不能促进有效的基因编辑。然而，供体质粒骨架中的额外切口显著提高了基因编辑效率。SNGD介导的基因编辑导致的indel频率明显低于DSB介导的方法。我们还表明，SNGD促进了人类细胞内源基因座的基因编辑。从机制上讲，SNGD介导的基因编辑需要靶基因和修复模板之间的长序列同源性，但不需要CtIP、RAD 51或RAD 52。因此，认为非经典同源性定向修复调节SNGD介导的基因编辑。总之，SNGD促进了精确和有效的基因编辑，可能是开发新基因治疗方法的一种有前途的策略。
CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.
登录
查看更多内容
影响因子：
46.9
作者：
Tsai, Shengdar Q.;Zheng, Zongli;Nguyen, Nhu T.;Liebers, Matthew;Topkar, Ved V.;Thapar, Vishal;Wyvekens, Nicolas;Khayter, Cyd;Iafrate, A. John;Le, Long P.;Aryee, Martin J.;Joung, J. Keith
通讯作者：
Joung, J. Keith
影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
通过 CRISPR/Cas9 介导的同源独立靶向整合进行体内基因组编辑
影响因子：
64.8
作者：
Suzuki K;Tsunekawa Y;Hernandez-Benitez R;Wu J;Zhu J;Kim EJ;Hatanaka F;Yamamoto M;Araoka T;Li Z;Kurita M;Hishida T;Li M;Aizawa E;Guo S;Chen S;Goebl A;Soligalla RD;Qu J;Jiang T;Fu X;Jafari M;Esteban CR;Berggren WT;Lajara J;Nuñez-Delicado E;Guillen P;Campistol JM;Matsuzaki F;Liu GH;Magistretti P;Zhang K;Callaway EM;Zhang K;Belmonte JC
通讯作者：
Belmonte JC
影响因子：
46.9
作者：
通讯作者：
--