RNA 引导的 Cas9 核酸酶的 DNA 靶向特异性。
DNA targeting specificity of RNA-guided Cas9 nucleases.
机构信息
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. [4].
出版信息
Nat Biotechnol. 2013 Sep;31(9):827-32. doi: 10.1038/nbt.2647. Epub 2013 Jul 21.
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
化脓性链球菌 Cas9（SpCas9）核酸酶可以通过单指导 RNA（sgRNA）有效地靶向基因组位点，从而实现基因组编辑。在这里，我们描述了 SpCas9 在人细胞中的靶向特异性，以指导靶位点的选择并避免脱靶效应。我们的研究评估了超过 700 种向导 RNA 变体和 SpCas9 诱导的在 293T 和 293FT 细胞中超过 100 个预测的基因组脱靶位点的缺失突变水平。我们发现 SpCas9 以序列依赖性的方式容忍向导 RNA 和靶 DNA 之间在不同位置的错配，对错配的数量、位置和分布敏感。我们还表明，SpCas9 介导的切割不受 DNA 甲基化的影响，并且可以滴定 SpCas9 和 sgRNA 的剂量以最小化脱靶修饰。为了促进哺乳动物基因组工程应用，我们提供了一个基于网络的软件工具，用于指导靶序列的选择和验证以及脱靶分析。
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