Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases.
Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases.
复制标题
人多能干细胞中G2/M细胞周期阶段的富集增强了可自定义的内切酶的HDR介导的基因修复。
DOI：
10.1038/srep21264
复制
发表时间：
2016-02-18
影响因子：
4.6
通讯作者：
Borowiak M
中科院分区：
文献类型：
--
作者：
Yang D;Scavuzzo MA;Chmielowiec J;Sharp R;Bajic A;Borowiak M
高效的基因编辑对于在再生医学中充分利用人类多能干细胞（hPSC）至关重要。定制的基于核酸内切酶的基因靶向涉及两种DNA修复机制：同源定向修复（HDR）和非同源末端连接（NHEJ）。HDR是常见应用如敲入、敲除或精确诱变的优选机制，但在hPSC中仍然效率低下。在这里，我们证明了使处于G2/M期的hPSC与ABT期同步化使靶向基因编辑（定义为正确的靶向盒整合）增加了3至6倍。我们观察到使用ZFN、TALEN、两种CRISPR/Cas9和CRISPR/Cas9切口酶靶向三种hPSC系中的五种基因的效率提高：三种人胚胎干细胞系、神经祖细胞和糖尿病iPSC。神经祖细胞和糖尿病iPSC。可逆同步化对多能性或分化没有影响。靶向基因编辑的增加与基因座无关，并且对细胞周期阶段具有特异性，因为与G1期细胞相比，G2/M期富集细胞的靶向效率增加了6倍。同时用SCR 7抑制NHEJ不会增加HDR或进一步提高基因靶向效率，表明HR是G2/M期阻滞后的主要DNA修复机制。本文概述的方法使hPSC中的基因编辑成为疾病建模，再生医学和基于细胞的治疗的更可行的工具。
Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies.