Consequences of Cas9 cleavage in the chromosome of Escherichia coli.
Consequences of Cas9 cleavage in the chromosome of Escherichia coli.
复制标题
DOI：
10.1093/nar/gkw223
复制
发表时间：
2016-05-19
影响因子：
14.9
通讯作者：
Bikard D
中科院分区：
文献类型：
--
作者：
Cui L;Bikard D
CRISPR-Cas 系统中的 RNA 引导 Cas9 核酸酶已成为一种强大的生物技术工具。 Cas9 的特异性可以通过重新编程来切割细胞染色体中所需的序列，只需改变小向导 RNA 的序列即可。与大多数真核生物不同，据报道，细菌染色体中的 Cas9 裂解会杀死细胞。然而，细胞死亡的机制仍有待研究。细菌主要依靠与姐妹染色体的同源重组（HR）来修复双链断裂。在这里，我们发现大肠杆菌染色体同一位置的所有拷贝同时裂解无法修复，从而导致细胞死亡。然而，通过 HR 途径的持续修复，可以容忍低效的裂解。为了可靠地杀死细胞，可以使用 Mu 噬菌体 Gam 蛋白来阻断 HR。最后，从结核分枝杆菌引入非同源末端连接（NHEJ）途径并不能将细胞从Cas9介导的杀伤中拯救出来，但确实以低频率引入了小缺失。这项工作让我们更好地了解 Cas9 切割细菌染色体的后果，这将有助于未来 CRISPR 工具的开发。
The RNA-guided Cas9 nuclease from CRISPR-Cas systems has emerged as a powerful biotechnological tool. The specificity of Cas9 can be reprogrammed to cleave desired sequences in a cell's chromosome simply by changing the sequence of a small guide RNA. Unlike in most eukaryotes, Cas9 cleavage in the chromosome of bacteria has been reported to kill the cell. However, the mechanism of cell death remains to be investigated. Bacteria mainly rely on homologous recombination (HR) with sister chromosomes to repair double strand breaks. Here, we show that the simultaneous cleavage of all copies of the Escherichia coli chromosome at the same position cannot be repaired, leading to cell death. However, inefficient cleavage can be tolerated through continuous repair by the HR pathway. In order to kill cells reliably, HR can be blocked using the Mu phage Gam protein. Finally, the introduction of the non-homologous end joining (NHEJ) pathway from Mycobacterium tuberculosis was not able to rescue the cells from Cas9-mediated killing, but did introduce small deletions at a low frequency. This work provides a better understanding of the consequences of Cas9 cleavage in bacterial chromosomes which will be instrumental in the development of future CRISPR tools.