Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.
Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity.
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DOI：
10.1093/nar/gkad170
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发表时间：
2023-04-24
影响因子：
14.9
通讯作者：
Bikard, David
中科院分区：
文献类型：
--
作者：
Rostain, William;Grebert, Theophile;Vyhovskyi, Danylo;Pizarro, Paula Thiel;Tshinsele-Van Bellingen, Gatwa;Cui, Lun;Bikard, David
源自 Cas9 RNA 引导核酸酶的遗传工具为研究和改造细菌提供了必要的能力。尽管在 Cas9 应用于哺乳动物细胞的早期就注意到了脱靶效应的重要性，但由于细菌基因组较小，Cas9 的脱靶切割很容易避免。尽管如此，一些研究报告了 Cas9 表达有毒的实验设置，即使使用 Cas9 的催化死亡变体 (dCas9) 也是如此。具体来说，dCas9 在与共享特定 PAM（原型间隔子相邻基序）-近端序列基序的指导 RNA 复合时被证明是有毒的。在这里，我们证明这种毒性是由 Cas9 与必需基因启动子的脱靶结合引起的，其中 PAM 近端序列中的同一性只有 4 nt 的脱靶基因就会发生沉默。在大肠杆菌和其他肠细菌的各种菌株中进行的筛选表明，毒性指导 RNA 的性质随着脱靶位置序列的进化而变化。这些结果凸显了 Cas9 可能与细菌基因组中数百个脱靶位置结合，从而导致不良影响。在细菌 CRISPR-Cas 实验的设计和解释中必须考虑到这种现象。
Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While the importance of off-target effects was noted early in Cas9’s application to mammalian cells, off-target cleavage by Cas9 in bacterial genomes is easily avoided due to their smaller size. Despite this, several studies have reported experimental setups in which Cas9 expression was toxic, even when using the catalytic dead variant of Cas9 (dCas9). Specifically, dCas9 was shown to be toxic when in complex with guide RNAs sharing specific PAM (protospacer adjacent motif)-proximal sequence motifs. Here, we demonstrate that this toxicity is caused by off-target binding of Cas9 to the promoter of essential genes, with silencing of off-target genes occurring with as little as 4 nt of identity in the PAM-proximal sequence. Screens performed in various strains of Escherichia coli and other enterobacteria show that the nature of toxic guide RNAs changes together with the evolution of sequences at off-target positions. These results highlight the potential for Cas9 to bind to hundreds of off-target positions in bacterial genomes, leading to undesired effects. This phenomenon must be considered in the design and interpretation of CRISPR–Cas experiments in bacteria.
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影响因子：
16.6
作者：
Collias D;Beisel CL
通讯作者：
Beisel CL
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
3.8
作者：
Luo ML;Leenay RT;Beisel CL
通讯作者：
Beisel CL
DOI：
10.1126/science.aah7084
发表时间：
2017-09-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Jones DL;Leroy P;Unoson C;Fange D;Ćurić V;Lawson MJ;Elf J
通讯作者：
Elf J
影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR