Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.
Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression.
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通过瞬时 BCL-XL 过表达，在人类多能干细胞中通过 CRISPR-Cas9 实现高效基因组编辑
DOI：
10.1093/nar/gky804
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发表时间：
2018-11-02
影响因子：
14.9
通讯作者：
Zhang XB
中科院分区：
文献类型：
--
作者：
Li XL;Li GH;Fu J;Fu YW;Zhang L;Chen W;Arakaki C;Zhang JP;Wen W;Zhao M;Chen WV;Botimer GD;Baylink D;Aranda L;Choi H;Bechar R;Talbot P;Sun CK;Cheng T;Zhang XB
人类诱导多能干细胞（iPSC）的基因组编辑有助于功能基因组学，疾病建模和再生医学。然而，低编辑效率阻碍了CRISPR-Cas9技术在创建敲入（KI）或敲除（KO）iPSC系中的应用，这主要是由于用编辑质粒电穿孔后的大量细胞死亡。在这里，我们报告了瞬时递送BCL-XL在质粒转染后使iPSC存活率增加10倍，导致同源定向修复（HDR）KI效率增加20至100倍，非同源末端连接（NHEJ）KO效率增加5倍。用BCL抑制剂ABT-263治疗进一步将HDR效率提高了70%，KO效率提高了40%。增加的基因组编辑效率归因于电穿孔后存活细胞中Cas9和sgRNA的更高表达。HDR或NHEJ效率达到95%，其中双重编辑，然后选择具有选择性基因的HDR插入的细胞。此外，可以在具有双等位基因HDR KO的大量细胞群体中实现100%的KO效率，随后进行双重选择，从而消除了单细胞克隆的必要性。总之，这些简单而高效的编辑策略为从操纵人类iPSC基因组到创建基因修饰动物模型的应用提供了有用的工具。
Genome editing of human induced pluripotent stem cells (iPSCs) is instrumental for functional genomics, disease modeling, and regenerative medicine. However, low editing efficiency has hampered the applications of CRISPR–Cas9 technology in creating knockin (KI) or knockout (KO) iPSC lines, which is largely due to massive cell death after electroporation with editing plasmids. Here, we report that the transient delivery of BCL-XL increases iPSC survival by ∼10-fold after plasmid transfection, leading to a 20- to 100-fold increase in homology-directed repair (HDR) KI efficiency and a 5-fold increase in non-homologous end joining (NHEJ) KO efficiency. Treatment with a BCL inhibitor ABT-263 further improves HDR efficiency by 70% and KO efficiency by 40%. The increased genome editing efficiency is attributed to higher expressions of Cas9 and sgRNA in surviving cells after electroporation. HDR or NHEJ efficiency reaches 95% with dual editing followed by selection of cells with HDR insertion of a selective gene. Moreover, KO efficiency of 100% can be achieved in a bulk population of cells with biallelic HDR KO followed by double selection, abrogating the necessity for single cell cloning. Taken together, these simple yet highly efficient editing strategies provide useful tools for applications ranging from manipulating human iPSC genomes to creating gene-modified animal models.
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影响因子：
16.6
作者：
Chen X;Janssen JM;Liu J;Maggio I;'t Jong AEJ;Mikkers HMM;Gonçalves MAFV
通讯作者：
Gonçalves MAFV
BCl-XL增强了人类干细胞的单细胞存活和扩展，而不会影响自我更新。
影响因子：
1.2
作者：
Bai, Hao;Chen, Kang;Gao, Yong-Xing;Arzigian, Melanie;Xie, Yin-Liang;Malcosky, Christopher;Yang, Yong-Guang;Wu, Wen-Shu;Wang, Zack Z.
通讯作者：
Wang, Zack Z.
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
16
作者：
Dumitru, Raluca;Gama, Vivian;Fagan, B. Matthew;Bower, Jacquelyn J.;Swahari, Vijay;Pevny, Larysa H.;Deshmukh, Mohanish
通讯作者：
Deshmukh, Mohanish
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F