High-Level Precise Knockin of iPSCs by Simultaneous Reprogramming and Genome Editing of Human Peripheral Blood Mononuclear Cells.
High-Level Precise Knockin of iPSCs by Simultaneous Reprogramming and Genome Editing of Human Peripheral Blood Mononuclear Cells.
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通过人外周血单核细胞的同时重编程和基因组编辑对 iPSC 进行高水平精确敲入
DOI：
10.1016/j.stemcr.2018.04.013
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发表时间：
2018-06-05
影响因子：
5.9
通讯作者：
Zhang XB
中科院分区：
文献类型：
--
作者：
Wen W;Cheng X;Fu Y;Meng F;Zhang JP;Zhang L;Li XL;Yang Z;Xu J;Zhang F;Botimer GD;Yuan W;Sun C;Cheng T;Zhang XB
我们已经开发了一种改进的附加型载体系统，用于从外周血单核细胞有效产生无整合的诱导多能干细胞（iPSC）。最近，我们报道了使用优化的CRISPR-Cas9系统与双切供体一起将同源定向修复介导的精确基因敲入效率提高了5至10倍。在这里，我们报告了血细胞重编程和基因组编辑在一个步骤中的整合。我们发现使用单一载体表达Cas9和KLF 4显著提高了基因组编辑效率，并且添加SV 40 LT进一步提高了敲入效率。在这些优化之后，可以在没有任何选择的情况下在批量iPSC群体中实现高达40%的基因组编辑效率。大多数编辑的细胞显示出iPSC的特征和基因组完整性。我们改进的方法整合了重编程和基因组编辑，应该可以加快精准和再生医学的基础研究和临床应用。共表达Cas9和KLF 4提高了重编程iPSCs的编辑效率SV 40 LT的包含进一步提高了敲入效率大多数基因编辑的细胞显示出iPSCs的特征和基因组完整性在这篇文章中，Zhang博士和同事描述了一种附加型载体系统，用于从外周血单核细胞（PB MNC）一步生成和基因组编辑（敲入）无整合的iPSCs。通过在单个载体中共表达Cas9和KLF 4并添加SV 40 LT，可以在批量iPSC中实现高达40%的敲入编辑效率。
We have developed an improved episomal vector system for efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells. More recently, we reported that the use of an optimized CRISPR-Cas9 system together with a double-cut donor increases homology-directed repair-mediated precise gene knockin efficiency by 5- to 10-fold. Here, we report the integration of blood cell reprogramming and genome editing in a single step. We found that expression of Cas9 and KLF4 using a single vector significantly increases genome editing efficiency, and addition of SV40LT further enhances knockin efficiency. After these optimizations, genome editing efficiency of up to 40% in the bulk iPSC population can be achieved without any selection. Most of the edited cells show characteristics of iPSCs and genome integrity. Our improved approach, which integrates reprogramming and genome editing, should expedite both basic research and clinical applications of precision and regenerative medicine. Co-expressing Cas9 and KLF4 increases editing efficiency in reprogrammed iPSCs Inclusion of SV40LT further increases knockin efficiency Most of the gene edited cells show characteristics of iPSCs and genome integrity In this article, Dr. Zhang and colleagues describe an episomal vector system for one-step generation and genome editing (knockin) of integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). Up to 40% knockin editing efficiencies in bulk iPSCs can be achieved by co-expression of Cas9 and KLF4 in a single vector and with addition of SV40LT.
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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
DOI：
10.7534/j.issn.1009-2137.2014.03.002
发表时间：
2014-06-01
期刊：
Zhongguo shi yan xue ye xue za zhi
影响因子：
--
作者：
Liu, Shu-Ping;Li, Yan-Xin;Yuan, Wei-Ping
通讯作者：
Yuan, Wei-Ping
DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Irion U;Krauss J;Nüsslein-Volhard C
通讯作者：
Nüsslein-Volhard C
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
7
作者：
Kim S;Kim D;Cho SW;Kim J;Kim JS
通讯作者：
Kim JS