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 induction³. 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 mutations¹⁴, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

中文翻译：

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p53抑制人多能干细胞中的CRISPR-Cas9工程。

CRISPR / Cas9彻底改变了我们在人类细胞^1-3中进行基因组工程和进行全基因组筛选的能力。尽管某些细胞类型适合进行基因组工程改造，但人类多能干细胞（hPSC）的基因组却难以进行工程改造，相对于肿瘤细胞系或小鼠胚胎干细胞而言，其效率降低了^3-13。在这里，使用具有稳定整合Cas9或瞬时递送Cas9-核糖核蛋白（RNP）的hPSC品系，我们实现了平均插入或缺失（插入/缺失）效率大于80％。这种indel生成的高效率表明，Cas9诱导的双链断裂（DSB）具有毒性，并杀死了大多数hPSC。在先前的研究中，Cas9在hPSC中的毒性不太明显，因为其转染效率低，随后DSB诱导率较低³。对DSB的毒性反应是P53 / TP53依赖性的，从而严重降低了具有野生型P53基因的hPSC中精确基因组工程的效率。我们的结果表明，Cas9毒性为CRISPR / Cas9在hPSCs中的基因组工程和筛选中高通量使用创造了障碍。此外，由于hPSC可以获取P53突变¹⁴，因此应谨慎使用CRISPR / Cas9改造的hPSC进行细胞替代疗法，并应监测此类改造的hPSC的P53功能。