Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.
Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.
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DOI：
10.1016/j.cell.2016.12.009
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发表时间：
2017-01-12
期刊：
影响因子：
64.5
通讯作者：
Bondy-Denomy J
中科院分区：
文献类型：
--
作者：
Rauch BJ;Silvis MR;Hultquist JF;Waters CS;McGregor MJ;Krogan NJ;Bondy-Denomy J
细菌 CRISPR-Cas 系统利用序列特异性 RNA 引导的核酸酶来防御噬菌体感染。作为一种对策，已知许多噬菌体会产生蛋白质来阻断 1 类 CRISPR-Cas 系统的功能。然而，目前尚无已知蛋白质能够抑制广泛使用的 2 类 CRISPR-Cas9 系统。为了找到这些抑制剂，我们搜索了包含 cas9 的细菌基因组，寻找 CRISPR 间隔区及其靶标的共存，这是 CRISPR 抑制的潜在指标。该分析发现了由单核细胞增生李斯特氏菌原噬菌体编码的四种独特的 II-A 型 CRISPR-Cas9 抑制剂蛋白。超过一半带有 cas9 的单核细胞增生李斯特菌菌株至少含有一种原噬菌体编码的抑制剂，表明 CRISPR-Cas9 广泛失活。在大肠杆菌和人类细胞中进行检测时，其中两种抑制剂还可阻断广泛使用的化脓性链球菌 Cas9。这些天然 Cas9 特异性“抗 CRISPR”提供了可用于调节 CRISPR-Cas9 基因组工程活性的工具。由单核细胞增生李斯特氏菌原噬菌体编码的四种 CRISPR-Cas9 抑制剂蛋白可阻止细菌和人类细胞中的 Cas9 结合和基因编辑，包括目前使用最广泛的来自化脓性链球菌的 Cas9。
Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a counter-measure, numerous phages are known that produce proteins to block the function of Class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used Class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific “anti-CRISPRs” present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9. Four CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages prevent Cas9 binding and gene editing in bacteria and human cells, including currently the most widely used Cas9 from Streptococcus pyogenes.