p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells
p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells
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DOI：
10.1038/s41591-018-0050-6
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发表时间：
2018-07-01
期刊：
影响因子：
82.9
通讯作者：
Kaykas, Ajamete
中科院分区：
文献类型：
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作者：
Ihry, Robert J.;Worringer, Kathleen A.;Kaykas, Ajamete
CRISPR/Cas9 彻底改变了我们在人类细胞中设计基因组和进行全基因组筛选的能力(1-3)。虽然某些细胞类型适合进行基因组工程，但人类多能干细胞 (hPSC) 的基因组一直难以工程化，相对于肿瘤细胞系或小鼠胚胎干细胞，其效率较低(3-13)。在这里，使用稳定整合 Cas9 或瞬时递送 Cas9-核糖核蛋白 (RNP) 的 hPSC 细胞系，我们实现了大于 80% 的平均插入或缺失 (indel) 效率。这种高效的 indel 生成表明 Cas9 诱导的双链断裂 (DSB) 具有毒性，会杀死大多数 hPSC。在之前的研究中，由于转染效率低以及随后的 DSB 诱导率低，Cas9 在 hPSC 中的毒性不太明显 (3)。 DSB 的毒性反应是 P53/TP53 依赖性的，因此在具有野生型 P53 基因的 hPSC 中进行精确基因组工程的效率严重降低。我们的结果表明，Cas9 毒性对高通量使用 CRISPR/Cas9 在 hPSC 中进行基因组工程和筛选造成了障碍。此外，由于 hPSC 可以获得 P53 突变 (14)，因此使用 CRISPR/Cas9 工程化 hPSC 的细胞替代疗法应谨慎进行，并且应监测此类工程化 hPSC 的 P53 功能。
CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells(1-3). Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells(3-13). Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction(3). The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations(14), cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.