Internal guide RNA interactions interfere with Cas9-mediated cleavage.
Internal guide RNA interactions interfere with Cas9-mediated cleavage.
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DOI：
10.1038/ncomms11750
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发表时间：
2016-06-10
影响因子：
16.6
通讯作者：
Schier AF
中科院分区：
文献类型：
--
作者：
Thyme SB;Akhmetova L;Montague TG;Valen E;Schier AF
CRISPR/Cas系统使用指导RNA（gRNA）来指导序列特异性DNA切割。并不是每一种gRNA的酶切和控制gRNA活性的机制都还没有解决。低活性可能是由于未能形成功能性Cas9-gRNA复合物或不能在体内识别靶标。在这里，我们通过比较斑马鱼胚胎中的诱变率与体外切割试验表明，这两种现象都会影响Cas9的活性。在体内，我们的研究结果表明，基因组因子，如CTCF抑制诱变。比较具有不同体外活性的几乎相同的gRNA序列揭示了内部gRNA相互作用减少切割。即使含有这些结构的gRNA不产生有切割能力的复合物，它们也可以与活性gRNA竞争结合Cas9。这些结果表明，基因组背景和内部gRNA相互作用都可以干扰Cas9介导的切割，并阐明Cas9-gRNA复合物形成的先前未表征的特征。 虽然CRISPR-Cas9系统已经彻底改变了分子生物学，但为什么不是每一个引导RNA都能靶向DNA切割仍然是一个谜。在这里，作者表明基因组背景和内部gRNA相互作用可以抑制切割。
The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation. While the CRISPR-Cas9 system has revolutionised molecular biology, it is still a mystery why not every guide RNA elicits target DNA cleavage. Here the authors show that genomic context and internal gRNA interactions can inhibit cleavage.