具有更高特异性的理性设计的Cas9核酸酶。
Rationally engineered Cas9 nucleases with improved specificity.
作者信息
Slaymaker Ian M, Gao Linyi, Zetsche Bernd, Scott David A, Yan Winston X, Zhang Feng
机构信息
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Science. 2016 Jan 1;351(6268):84-8. doi: 10.1126/science.aad5227. Epub 2015 Dec 1.
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
RNA引导的核酸内切酶Cas9是一种多功能的基因组编辑工具，具有从治疗到基因功能注释等广泛的应用。Cas9在与短RNA引导序列互补的靶向基因组位点产生双链断裂（DSB）。然而，Cas9可切割与引导序列不完全互补的脱靶位点，这给基因组编辑带来了重大挑战。在此，我们利用结构引导的蛋白质工程来提高化脓性链球菌Cas9（SpCas9）的特异性。通过靶向深度测序和无偏全基因组脱靶分析来评估Cas9介导的人类细胞中的DNA切割，我们证明“增强特异性”的SpCas9（eSpCas9）变体减少了脱靶效应并维持了强大的靶向切割能力。因此，eSpCas9对于需要高度特异性的基因组编辑应用可能具有广泛的用途。
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