The spacer size of I-B CRISPR is modulated by the terminal sequence of the protospacer.
The spacer size of I-B CRISPR is modulated by the terminal sequence of the protospacer.
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I-B CRISPR 的间隔区大小由原型间隔区的末端序列调节。
DOI：
10.1093/nar/gkx229
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发表时间：
2017-05-05
影响因子：
14.9
通讯作者：
Xiang H
中科院分区：
文献类型：
--
作者：
Li M;Gong L;Zhao D;Zhou J;Xiang H
原核生物通过将外来DNA作为间隔物加入CRISPR阵列来记忆入侵者的信息。虽然间隔区的大小已被建议由获取复合体的架构预先定义，但通常存在意想不到的异质性。在这里，我们探索了这种异质性的原因在Haloarcula hispanica I-B CRISPR。适应分析后的高通量测序显示37957个新间隔区之间存在显著的大小差异，这似乎是序列依赖的。始终如一的是，间隔区3PAM末端的第三个核苷酸(΄-远端)显示出明显的对胞嘧啶的偏爱，在原间隔区序列中突变这个胞嘧啶可以改变最终的间隔区大小。此外，导致大部分观察到的΄(Protspacer相邻基序)不准确的5个蛋白质末端(PAM-End)的滑移也倾向于改变间隔子的大小。我们认为PAM-Protspacer序列的两端应该表现出核苷酸的选择性(具有不同的严谨性)，这在一定程度上微调了结构尺子，以指定间隔子的大小。
Prokaryotes memorize invader information by incorporating alien DNA as spacers into CRISPR arrays. Although the spacer size has been suggested to be predefined by the architecture of the acquisition complex, there is usually an unexpected heterogeneity. Here, we explored the causes of this heterogeneity in Haloarcula hispanica I-B CRISPR. High-throughput sequencing following adaptation assays demonstrated significant size variation among 37 957 new spacers, which appeared to be sequence-dependent. Consistently, the third nucleotide at the spacer 3΄-end (PAM-distal end) showed an evident bias for cytosine and mutating this cytosine in the protospacer sequence could change the final spacer size. In addition, slippage of the 5΄-end (PAM-end), which contributed to most of the observed PAM (protospacer adjacent motif) inaccuracy, also tended to change the spacer size. We propose that both ends of the PAM-protospacer sequence should exhibit nucleotide selectivity (with different stringencies), which fine-tunes the structural ruler, to a certain extent, to specify the spacer size.