Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.
Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.
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DOI：
10.1093/nar/gkx097
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发表时间：
2017-04-07
影响因子：
14.9
通讯作者：
Savitskaya E
中科院分区：
文献类型：
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作者：
Musharova O;Klimuk E;Datsenko KA;Metlitskaya A;Logacheva M;Semenova E;Severinov K;Savitskaya E
在引发的 CRISPR 适应过程中，优先从 CRISPR 干扰机制识别的 DNA 中选择间隔区，在 I 型 CRISPR-Cas 系统中，间隔区由定位互补靶点的 CRISPR RNA (crRNA) 结合效应子级联复合物和 Cas3 执行核酸酶/解旋酶组成。 Cas1 和 Cas2 蛋白的复合物能够在 CRISPR 阵列中插入新的间隔区。在这里，我们发现，在经历引发适应的大肠杆菌细胞中，间隔大小的外源 DNA 片段与 Cas1 相关。基于对核酸酶消化的敏感性，相关 DNA 不处于标准双链状态。以 Cas1 和 Cas3 依赖性方式从外源 DNA 的一条链上切下间隔子大小的片段。这些片段是由更长的 S1 核酸酶敏感外源 DNA 片段生成的，需要 Cas3 才能生成。我们提出，在 CRISPR 干扰过程中，Cas3 会产生外源 DNA 片段，这些片段可以被 Cas1-Cas2 适应复合体识别，该复合体切除间隔区大小的片段并将其引导至插入 CRISPR 阵列中。
During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR–Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1–Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.