Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, BIOL 1020, Cambridge, Massachusetts 02138, USA.
Computational Biology Unit, Department of Informatics, University of Bergen, 5020 Bergen, Norway.
Nat Commun. 2016 Jun 10;7:11750. doi: 10.1038/ncomms11750.
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.
CRISPR/Cas 系统使用向导 RNA（gRNA）来指导序列特异性 DNA 切割。并非每个 gRNA 都能引发切割，并且调控 gRNA 活性的机制尚未解决。活性低可能是由于未能形成功能性 Cas9-gRNA 复合物，或者无法在体内识别靶标。通过比较斑马鱼胚胎中的突变率和体外切割实验，我们发现这两种现象都会影响 Cas9 的活性。在体内，我们的结果表明，基因组因素（如 CTCF）会抑制突变。比较具有不同体外活性的近乎相同的 gRNA 序列表明，gRNA 内部相互作用会降低切割效率。尽管含有这些结构的 gRNA 不能产生具有切割活性的复合物，但它们可以与活性 gRNA 竞争 Cas9 的结合。这些结果表明，基因组背景和 gRNA 内部相互作用都可能干扰 Cas9 介导的切割，并阐明 Cas9-gRNA 复合物形成的以前未表征的特征。
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