In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.
In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.
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DOI：
10.1038/s41467-017-00687-1
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发表时间：
2017-09-22
影响因子：
16.6
通讯作者：
Gonçalves MAFV
中科院分区：
文献类型：
--
作者：
Chen X;Janssen JM;Liu J;Maggio I;'t Jong AEJ;Mikkers HMM;Gonçalves MAFV
精确的基因组编辑涉及供体DNA和染色体序列之间的同源重组，这些序列受到可编程核酸酶造成的双链DNA断裂的影响。理想情况下，基因组编辑应该是高效、具体和准确的。然而，除了构成潜在的易位启动损伤外，双链DNA断裂(靶向或其他)大多通过不可预测和突变的非同源重组过程修复。在这里，我们报告了基于CRISPR-Cas9组件的RNA引导的核酸酶在供体质粒和染色体靶点协调形成成对的单链DNA断裂或NICK，触发了对包括多能干细胞在内的人类细胞中大的遗传有效载荷的无缝同源导向的基因靶向。重要的是，除了显著降低基因组修改过程的致突变性外，这种反式配对划痕策略还实现了多路、单步、基因靶向，与标准的双链DNA断裂依赖方法相比，产生了更高频率的准确编辑细胞。基于CRISPR-Cas9的基因编辑涉及目标序列的双链断裂，通常通过诱变的非同源末端连接来修复。在这里，作者使用Cas9昵称酶在供体和目标DNA中产生协调的单链断裂，以进行精确的同源定向基因编辑。
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach. CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
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影响因子：
4.6
作者：
Maggio I;Holkers M;Liu J;Janssen JM;Chen X;Gonçalves MA
通讯作者：
Gonçalves MA
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：
11.2
作者：
Mendell, Jerry R.;Shilling, Chris;Weiss, Robert B.
通讯作者：
Weiss, Robert B.
影响因子：
2.9
作者：
Dambrot, C.;van de Pas, S.;Freund, C.
通讯作者：
Freund, C.
影响因子：
14.9
作者：
He X;Tan C;Wang F;Wang Y;Zhou R;Cui D;You W;Zhao H;Ren J;Feng B
通讯作者：
Feng B